Peptide-containing linkers for antibody-drug conjugates

ABSTRACT

The present disclosure relates generally to antibody-drug conjugates comprising peptide-containing linkers and to methods of using these conjugates as therapeutics and/or diagnostics. Also disclosed herein are peptide-containing scaffolds useful to conjugate with a targeting moiety (e.g., an antibody), a drug, or both to produce the antibody-drug conjugates.

RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S.provisional application Nos. 62/425,895, filed Nov. 23, 2016 and62/572,010, filed Oct. 13, 2017, under 35 USC § 119(e). The content ofthese applications are hereby incorporated by reference in theirentirety.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the file named“MRSN-019_001US_322140-2233_SeqListing_ST25.txt”, which was created onJan. 12, 2018, and is 4 KB in size are hereby incorporated by referencein their entirety.

BACKGROUND

Traditionally, pharmaceuticals have primarily consisted of smallmolecules that are dispensed orally (as solid pills and liquids) or asinjectables. Over the past three decades, formulations (i.e.,compositions that control the route and/or rate of drug delivery andallow delivery of the therapeutic agent at the site where it is needed)have become increasingly common and complex. Nevertheless, manyquestions and challenges regarding the development of new treatments aswell as the mechanisms with which to administer them remain to beaddressed. For example, many drugs exhibit limited or otherwise reducedpotencies and therapeutic effects because they are either generallysubject to partial degradation before they reach a desired target in thebody, or accumulate in tissues other than the target, or both.

One objective in the field of drug delivery systems, therefore, is todeliver medications intact to specifically targeted areas of the bodythrough a system that can stabilize the drug and control the in vivotransfer of the therapeutic agent utilizing either physiological orchemical mechanisms, or both.

Antibody-drug conjugates have been developed as target-specifictherapeutic agents. Antibodies against various cancer cell-surfaceantigens have been conjugated with different cytotoxic agents thatinhibit various essential cellular targets such as microtubules (such asmaytansinoids, auristatins, and taxanes, see, e.g., U.S. Pat. Nos.5,208,020; 5,416,064; 6,333,410; 6,441,163; 6,340,701; 6,372,738;6,436,931; 6,596,757; and 7,276,497); DNA (such as calicheamicin,doxorubicin, and CC-1065 analogs; see, e.g., U.S. Pat. Nos. 5,475,092;5,585,499; 5,846,545; 6,534,660; 6,756,397; and 6,630,579). Antibodyconjugates with some of these cytotoxic drugs are actively beinginvestigated in the clinic for cancer therapy (see, e.g., Ricart, A. D.,and Tolcher, A. W., 2007, Nature Clinical Practice, 4, 245-255; Krop etal., 2010, J. Clin. Oncol., 28, 2698-2704). However, existingantibody-drug conjugates have exhibited a few limitations. A majorlimitation is their inability to deliver a sufficient concentration ofdrug to the target site because of the limited number of targetedantigens and the relatively moderate cytotoxicity of cancer drugs likemethotrexate, daunorubicin, maytansinoids, taxanes, and vincristine. Oneapproach to achieving significant cytotoxicity is by linkage of a largenumber of drug molecules either directly or indirectly to the antibody.However such heavily modified antibodies often display impaired bindingto the target antigen and/or fast in vivo clearance from the bloodstream. Therefore, there is a need to improve the ability to deliver asufficient concentration of a drug to the target such that maximumcytotoxicity for the drug is achieved.

SUMMARY

The present disclosure features a targeting moiety-drug conjugate thatis biodegradable, biocompatible and exhibits high drug load as well asstrong binding to target antigen. For example, the targeting moiety is aprotein based recognition-molecule (PBRM). The present disclosure alsofeatures a peptide-containing scaffold useful to conjugate with a PBRM,a drug, or both, so as to obtain the targeting moiety-drug conjugate.

In one aspect, the disclosure relates to a conjugate comprising atargeting moiety and one or more Linker-Drug moieties covalently bondedto the targeting moiety, wherein

each Linker-Drug moiety includes a Multifunctional Linker that connectsthe targeting moiety to one or more Drug Units through intermediacy of aReleasable Assembly Unit for each Drug Unit, and connects a hydrophilicgroup to the Drug Units of each Linker-Drug moiety,

wherein the Releasable Assembly units are capable of releasing free drugin proximity to a target site targeted by the targeting moiety, and

wherein the Multifunctional Linker comprises a peptide moiety betweenthe targeting moiety and the hydrophilic group, wherein the peptidemoiety includes at least two amino acids.

In another aspect, the disclosure relates to a conjugate comprising atargeting moiety and one or more Linker-Drug moieties covalently bondedto the targeting moiety, wherein

each Linker-Drug moiety includes a Multifunctional Linker that connectsthe targeting moiety to one or more Drug Units through intermediacy of aReleasable Assembly Unit for each Drug Unit, and connects a polyalcoholor a derivative thereof to the Drug Units of each Linker-Drug moiety,

wherein the Releasable Assembly units are capable of releasing free drugin proximity to a target site targeted by the targeting moiety.

The disclosure also relates to a conjugate of Formula (I):

wherein

a₁ is an integer from 0 to 1;

a₂ is an integer from 1 to 3;

a₃ is an integer from 0 to 1;

a₄ is an integer from 1 to about 5;

a₅ is an integer from 1 to 3;

d₁₃ is an integer from 1 to about 14;

PBRM denotes a protein based recognition-molecule;

L^(P′) is a divalent linker moiety connecting the PBRM to M^(P); ofwhich the corresponding monovalent moiety L^(P) contains a functionalgroup W^(P) that is capable of forming a covalent bond with a functionalgroup of the PBRM;

M^(P) is a Stretcher unit;

L^(M) is a bond, or a trivalent or tetravalent linker, and when L^(M) isa bond, a₂ is 1, when L^(M) is trivalent linker, a₂ is 2, or when L^(M)is a tetravalent linker, a₂ is 3;

L³ is a carbonyl-containing moiety;

M^(A) comprises a peptide moiety that contains at least two amino acids;

T¹ is a hydrophilic group and the

between T¹ and M^(A) denotes direct or indirect attachment of T¹ andM^(A);

each occurrence of D is independently a therapeutic agent having amolecular weight≤about 5 kDa; and

each occurrence of L^(D) is independently a divalent linker moietyconnecting D to M^(A) and comprises at least one cleavable bond suchthat when the bond is broken, D is released in an active form for itsintended therapeutic effect.

In yet another aspect, the disclosure relates to a peptide-containingscaffold, being any of Formulae (II)-(IX):

wherein

a₁ is an integer from 0 to 1;

a₂ is an integer from 1 to 3;

a₃ is an integer from 0 to 1;

a₄ is an integer from 1 to about 5;

a₅ is an integer from 1 to 3;

d₁₃ is an integer from 1 to about 14;

PBRM denotes a protein based recognition-molecule;

L^(P′) is a divalent linker moiety connecting the PBRM to M^(P); ofwhich the corresponding monovalent moiety L^(P) contains a functionalgroup W^(P) that is capable of forming a covalent bond with a functionalgroup of the PBRM;

M^(P) is a Stretcher unit;

L^(M) is a bond, or a trivalent or tetravalent linker, and when L^(M) isa bond, a₂ is 1, when L^(M) is a trivalent linker, a₂ is 2, or whenL^(M) is a tetravalent linker, a₂ is 3;

L³ is a carbonyl-containing moiety;

M^(A) comprises a peptide moiety that contains at least two amino acids;

T¹ is a hydrophilic group and the

between T¹ and M^(A) denotes direct or indirect attachment of T¹ andM^(A);

each occurrence of W^(M) is independently hydrogen, a protecting group,a leaving group, or a functional group that is capable of connectingL^(M) to M^(P) by forming a covalent bond;

each occurrence of W^(D) is independently a functional group that iscapable of forming a covalent bond with a functional group of atherapeutic agent (“D”) having a molecular weight≤about 5 kDa; and

each occurrence of L^(D) is independently a divalent linker moietyconnecting W^(D) or D to M^(A) and L^(D) comprises at least onecleavable bond such that when the bond is broken, D is released in anactive form for its intended therapeutic effect.

The conjugates and scaffolds of the disclosure can include one or moreof the following features when applicable.

For example, each of the Drug Units and the hydrophilic group isconnected to the Multifunctional Linker in parallel orientation.

For example, the targeting moiety is a protein basedrecognition-molecule (PBRM). For example, the PBRM is an antibody orantibody fragment.

For example, the peptide moiety in the Multifunctional Linker includesfrom three to about sixteen amino acids, e.g., about 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or 16 amino acids.

For example, the peptide moiety in the Multifunctional Linker includesfrom three to about ten amino acids, e.g., about 4, 5, 6, 7, 8, 9 or 10amino acids.

For example, the peptide moiety contains from three to about ten aminoacids selected from glycine, serine, glutamic acid, aspartic acid,lysine, cysteine, a stereoisomer thereof (e.g., isoglutamic acid orisoaspartic acid), and a combination thereof.

For example, the peptide moiety contains at least four glycines and atleast one serine.

For example, the peptide moiety contains at least four glycines, atleast one serine and at least one glutamic acid or isoglutamic acid.

For example, the hydrophilic group comprises a polyalcohol or aderivative thereof, a polyether or a derivative thereof, or acombination thereof.

For example, the hydrophilic group comprises an amino polyalcohol, e.g.,glucamine or bis-glucamine.

For example, the hydrophilic group comprises:

For example, the hydrophilic group comprises:

For example, the amino polyalcohol is

in which

n₁ is an integer from 0 to about 6;

each R₅₈ is independently hydrogen or C₁₋₈ alkyl;

R₆₀ is a bond, a C₁₋₆ alkyl linker, or —CHR₅₉— in which R₅₉ is H, alkyl,cycloalkyl, or arylalkyl;

R₆₁ is CH₂OR₆₂, COOR₆₂, —(CH₂)_(n2)COOR₆₂, or a heterocycloalkylsubstituted with one or more hydroxyl;

R₆₂ is H or C₁₋₈ alkyl; and

n₂ is an integer from 1 to about 5.

For example, the hydrophilic group comprises

in which

n₄ is an integer from 1 to about 25;

each R₆₃ is independently hydrogen or C₁₋₈ alkyl;

R₆₄ is a bond or a C₁₋₈ alkyl linker;

R₆₅ is H, C₁₋₈ alkyl or —(CH₂)_(n2)COOR₆₂;

R₆₂ is H or C₁₋₈ alkyl; and

n₂ is an integer from 1 to about 5.

For example, the hydrophilic group comprises polyethylene glycol, e.g.,polyethylene glycol with about 6 to about 24 PEG subunits, preferablyabout 6 to about 12 PEG subunits or about 8 to about 12 PEG subunits.

For example, L³, when present, comprises —X—C₁₋₁₀ alkylene —C(O)—, withX directly connected to L^(M), in which X is CH₂, O, or NR₅, and R₅ ishydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈ cycloalkyl, COOH, or COO—C₁₋₆alkyl.

For example, L³, when present, is —NR₅—(CH₂)_(v)—C(O)— or—CH₂—(CH₂)_(v)—C(O)—NR₅—(CH₂)_(v)—C(O)—, in which each v independentlyis an integer from 1 to 10 (e.g., each v independently being an integerfrom 1 to 6, or from 2 to 4, or 2). For example, L³ is —NH—(CH₂)₂—C(O)—or —(CH₂)₂—C(O)—NH—(CH₂)₂—C(O)—.

For example, a₄ is 1, 2, or 3.

For example, d₁₃ is an integer from 1 to about 10, e.g., d₁₃ is 4 or 5.

For example, each W^(P), when present, is independently:

wherein

ring A is cycloalkyl or heterocycloalkyl;

ring B is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;

R^(1K) is a leaving group;

R^(1A) is a sulfur protecting group;

R^(1J) is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, orheterocycloalkyl moiety;

R^(2J) is hydrogen, an aliphatic, aryl, heteroaliphatic, or carbocyclicmoiety;

R^(3J) is C₁₋₆ alkyl and each of Z₁, Z₂, Z₃ and Z₇ is independently acarbon or nitrogen atom;

R^(4j) is hydrogen, halogen, OR, —NO₂, —CN, —S(O)₂R, C₁₋₂₄ alkyl (e.g.,C₁₋₆ alkyl), or 6-24 membered aryl or heteroaryl, wherein the C₁₋₂₄alkyl (e.g., C₁₋₆ alkyl), or 6-24 membered aryl or heteroaryl, isoptionally substituted with one or more aryl or heteroaryl; or twoR^(4j) together form an annelated cycloalkyl, heterocycloalkyl, aryl, orheteroaryl; R is hydrogen, alkyl, heteroalkyl, cycloalkyl, orheterocycloalkyl;

R^(5j) is C(R^(4j))₂, O, S or NR; and

z₁ is an integer 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

For example, R^(1K) is halo or RC(O)O— in which R is hydrogen, analiphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety. Forexample, R^(1A) is

in which r is 1 or 2 and each of R^(s1), R^(s2), and R^(s3) is hydrogen,an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

For example, ring A can be

wherein R^(6j) is hydrogen, halogen, C₁₋₂₄ alkyl (e.g., C₁₋₆ alkyl), or6-24 membered aryl or heteroaryl, wherein the C₁₋₂₄ alkyl (e.g., C₁₋₆alkyl), or 6-24 membered aryl or heteroaryl, is optionally substitutedwith one or more aryl or heteroaryl.

For example, ring A can be

For example, M^(P), when present, is —(Z₄)-[(Z₅)-(Z₆)]_(z)—, with Z₄connected to L^(P′) or L^(P) and Z₆ connected to L^(M); in which

z is 1, 2, or 3;

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to Z₅ or Z₆ when present or to L^(M) when Z₅ and Z₆ are bothabsent;

b₁ is an integer from 0 to 6;

e₁ is an integer from 0 to 8,

R₁₇ is C₁₋₁₀ alkylene, C₁₋₁₀ heteroalkylene, C₃₋₈ cycloalkylene, O—(C₁₋₈alkylene, arylene, —C₁₋₁₀ alkylene-arylene-, -arylene-C₁₋₁₀ alkylene-,—C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-, —(C₃₋₈ cycloalkylene-C₁₋₁₀alkylene-, 4 to 14-membered heterocycloalkylene, —C₁₋₁₀ alkylene-(4 to14-membered heterocycloalkylene)-, -(4 to 14-memberedheterocycloalkylene)-C₁₋₁₀ alkylene-, —C₁₋₁₀ alkylene-C(═O)—, —C₁₋₁₀heteroalkylene-C(═O)—, —C₃₋₈ cycloalkylene-C(═O)—, —O—(C₁₋₈alkyl)-C(═O)—, -arylene-C(═O)—, —C₁₋₁₀ alkylene-arylene-C(═O)—, -arylene—C₁₋₁₀ alkylene-C(═O)—, —C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-C(═O)—,—(C₃₋₈ cycloalkylene)-C₁₋₁₀ alkylene-C(═O)—, -4 to 14-memberedheterocycloalkylene-C(═O)—, —C₁₋₁₀ alkylene-(4 to 14-memberedheterocycloalkylene)-C(═O)—, -(4 to 14-memberedheterocycloalkylene)-C₁₋₁₀ alkylene-C(═O)—, —C₁₋₁₀ alkylene-NH—, —C₁₋₁₀heteroalkylene-NH—, —C₃₋₈ cycloalkylene-NH—, —O—(C₁₋₈ alkyl)-NH—,-arylene-NH—, —C₁₋₁₀ alkylene-arylene-NH—, -arylene-C₁₋₁₀ alkylene-NH—,—C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-NH—, —(C₃₋₈ cycloalkylene)-C₁₋₁₀alkylene-NH—, -4 to 14-membered heterocycloalkylene-NH—, —C₁₋₁₀alkylene-(4 to 14-membered heterocycloalkylene)-NH—, -(4 to 14-memberedheterocycloalkylene)-C₁₋₁₀ alkylene-NH—, —C₁₋₁₀ alkylene-S—,heteroalkylene-S—, —C₃₋₈ cycloalkylene-S—, —O—C₁₋₈ alkyl)-S—,-arylene-S—, —C₁₋₁₀ alkylene-arylene-S—, -arylene-C₁₋₁₀ alkylene-S—,alkylene-(C₃₋₈ cycloalkylene)-S—, —(C₃₋₈ cycloalkylene)-C₁₋₁₀alkylene-S—, -4 to 14-membered heterocycloalkylene-S—, —C₁₋₁₀alkylene-(4 to 14-membered heterocycloalkylene)-S—, or -(4 to14-membered heterocycloalkylene)-C₁-C₁₀ alkylene-S—;

each Z₅ independently is absent, R₅₇—R₁₇ or a polyether unit;

each R₅₇ independently is a bond, NR₂₃, S or O;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl; and

each Z₆ independently is absent, —C₁₋₁₀ alkyl-R₃—, —C₁₋₁₀ alkyl-NR₅—,—C₁₋₁₀ alkyl-C(O)—, —C₁₋₁₀ alkyl-O—, —C₁₋₁₀ alkyl-S— or —(C₁₋₁₀alkyl-R₃)_(g1)—C₁₋₁₀ alkyl-C(O)—;

each R₃ independently is —C(O)—NR₅— or —NR₅—C(O)—;

each R₅ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, COOH, or COO—C₁₋₆ alkyl; and

g₁ is an integer from 1 to 4.

For example, M^(P), when present, is

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to L^(M);

R₃ is —C(O)—NR₅ or —NR₅—C(O)—;

R₄ is a bond or —NR₅—(CR₂₀R₂₁)—C(O)—;

R₅ is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈ cycloalkyl, —COOH, or—COO—C₁₋₆ alkyl;

R₁₇ is C₁₋₁₀ alkylene, C₁₋₁₀ heteroalkylene, C₃₋₈ cycloalkylene, O—(C₁₋₈alkylene, arylene, —C₁₋₁₀ alkylene-arylene-, -arylene-C₁₋₁₀ alkylene-,—C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-, —(C₃₋₈ cycloalkylene-C₁₋₁₀alkylene-, 4 to 14-membered heterocycloalkylene, —C₁₋₁₀ alkylene-(4 to14-membered heterocycloalkylene)-, -(4 to 14-memberedheterocycloalkylene)-C₁₋₁₀ alkylene-, —C₁₋₁₀ alkylene-C(═O)—, —C₁₋₁₀heteroalkylene-C(═O)—, —C₃₋₈ cycloalkylene-C(═O)—, —O—(C₁₋₈alkyl)-C(═O)—, -arylene-C(═O)—, —C₁₋₁₀ alkylene-arylene-C(═O)—, -arylene—C₁₋₁₀ alkylene-C(═O)—, —C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-C(═O)—,—(C₃₋₈ cycloalkylene)-C₁₋₁₀ alkylene-C(═O)—, -4 to 14-memberedheterocycloalkylene-C(═O)—, —C₁₋₁₀ alkylene-(4 to 14-memberedheterocycloalkylene)-C(═O)—, -(4 to 14-memberedheterocycloalkylene)-C₁₋₁₀ alkylene-C(═O)—, —C₁₋₁₀ alkylene-NH—, —C₁₋₁₀heteroalkylene-NH—, —C₃₋₈ cycloalkylene-NH—, —O—(C₁₋₈ alkyl)-NH—,-arylene-NH—, alkylene-arylene-NH—, -arylene-C₁₋₁₀ alkylene-NH—, —C₁₋₁₀alkylene-(C₃₋₈ cycloalkylene)-NH—, —(C₃₋₈ cycloalkylene)-C₁₋₁₀alkylene-NH—, -4 to 14-membered heterocycloalkylene-NH—, alkylene-(4 to14-membered heterocycloalkylene)-NH—, -(4 to 14-memberedheterocycloalkylene)-C₁₋₁₀ alkylene-NH—, —C₁₋₁₀ alkylene-S—, —C₁₋₁₀heteroalkylene-S—, —C₃₋₈ cycloalkylene-S—, —O—C₁₋₈ alkyl)-S—,-arylene-S—, —C₁₋₁₀ alkylene-arylene-S—, -arylene-C₁₋₁₀ alkylene-S—,—C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-S—, —(C₃₋₈ cycloalkylene)-C₁₋₁₀alkylene-S—, -4 to 14-membered heterocycloalkylene-S—, —C₁₋₁₀alkylene-(4 to 14-membered heterocycloalkylene)-S—, or -(4 to14-membered heterocycloalkylene)-C₁-C₁₀ alkylene-S—;

each R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

each b₁ independently is an integer from 0 to 6;

e₁ is an integer from 0 to 8,

each f₁ independently is an integer from 1 to 6; and

g₂ is an integer from 1 to 4.

For example, M^(P), when present, is

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to L^(M).

For example, L^(M) is a bond and a₂ is 1.

For example, a₂ is 2 and L^(M) is

wherein

denotes attachment to M^(P) when present or attachment to L^(P) orL^(P′) when M^(P) is absent;

Y₁ denotes attachment to L³ when present or attachment to M^(A) when L³is absent;

R₂ and R′₂ are each independently hydrogen, an optionally substitutedC₁₋₆ alkyl, an optionally substituted C₂₋₆ alkenyl, an optionallysubstituted C₂₋₆ alkynyl, an optionally substituted C₃₋₁₉ branchedalkyl, an optionally substituted C₃₋₈ cycloalkyl, an optionallysubstituted C₆₋₁₀ aryl, an optionally substituted heteroaryl, anoptionally substituted C₁₋₆ heteroalkyl, C₁₋₆ alkoxy, aryloxy, C₁₋₆heteroalkoxy, C₂₋₆ alkanoyl, an optionally substituted arylcarbonyl,C₂₋₆ alkoxycarbonyl, C₂₋₆ alkanoyloxy, arylcarbonyloxy, an optionallysubstituted C₂₋₆ alkanoyl, an optionally substituted C₂₋₆ alkanoyloxy,an optionally substituted C₂₋₆ substituted alkanoyloxy, —COOH, or—COO—C₁₋₆ alkyl;

each of c₁, c₂, C₃, c₄, c₅, c₇, and c₈ is an integer independentlyranging between 0 and 10; and

each of d₁, d₂, d₃, d₄, d₅, and d₇ is an integer independently rangingbetween 0 and 10.

For example, a₂ is 2 and L^(M) is:

For example, a₂ is 3 and L^(M) is:

wherein:

denotes attachment to M^(P) when present or attachment to L^(P) orL^(P′) when M^(P) is absent;

Y₁ denotes attachment to L³ when present or attachment to M^(A) when L³is absent;

R₂ and R′₂ are each independently hydrogen, an optionally substitutedC₁₋₆ alkyl, an optionally substituted C₂₋₆ alkenyl, an optionallysubstituted C₂₋₆ alkynyl, an optionally substituted C₃₋₁₉ branchedalkyl, an optionally substituted C₃₋₈ cycloalkyl, an optionallysubstituted C₆₋₁₀ aryl, an optionally substituted heteroaryl, anoptionally substituted C₁₋₆ heteroalkyl, C₁₋₆ alkoxy, aryloxy, C₁₋₆heteroalkoxy, C₂₋₆ alkanoyl, an optionally substituted arylcarbonyl,C₂₋₆ alkoxycarbonyl, C₂₋₆ alkanoyloxy, arylcarbonyloxy, an optionallysubstituted C₂₋₆ alkanoyl, an optionally substituted C₂₋₆ alkanoyloxy,an optionally substituted C₂₋₆ substituted alkanoyloxy, —COOH, or—COO—C₁₋₆ alkyl;

each of c₁, c₂, c₃, c₄, c₅, c₆, c₇, and c₈ is an integer independentlyranging between 0 and 10;

each of d₁, d₂, d₃, d₄, d₅, d₆, d₇ and d₈ is an integer independentlyranging between 0 and 10; and

each of e₁, e₂, e₃, e₄, e₅, e₆, e₇, and e₈ is an integer independentlyranging between 0 and 10.

For example, a₂ is 3 and L^(M) is

For example, M^(A) comprises a peptide moiety that contains at leastabout five amino acids. For example, M^(A) comprises a peptide moietythat contains at most about sixteen amino acids, e.g., about 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids. For example, M^(A)comprises a peptide moiety that contains at most about ten amino acids,e.g., about 4, 5, 6, 7, 8, 9, or 10 amino acids.

For example, M^(A) comprises a peptide moiety that contains from threeto about ten amino acids selected from glycine, serine, glutamic acid,aspartic acid, lysine, cysteine, a stereoisomer thereof (e.g.,isoglutamic acid or isoaspartic acid), and a combination thereof.

For example, M^(A) comprises a peptide moiety that contains at leastfour glycines and at least one serine.

For example, M^(A) comprises a peptide moiety that contains at leastfour glycines and at least one glutamic acid.

For example, M^(A) comprises a peptide moiety that contains at leastfour glycines, at least one serine and at least one glutamic acid.

For example, the ratio between D and PBRM or the ratio between DrugUnits and the targeting moiety can be greater than 1:1, and up to 50:1,e.g., between 2:1 and 40:1; between 5:1 and 20:1; between 10:1 and 50:1,between 25:1 and 50:1, or between 30:1 and 50:1. Examples of PBRMinclude but are not limited to, full length antibodies such as IgG andIgM, antibody fragments such as Fabs, scFv, camelids, Fab2, and thelike, small proteins, and peptides.

For example, the ratio between D and PBRM or the ratio between DrugUnits and the targeting moiety is about 50:1, 40:1, 25:1, 20:1, 15:1,10:1, 9:1, 8:1, 7:1, 6;1, 5:1, 4:1, 3:1, or 2:1.

For example, the ratio between D and PBRM or the ratio between DrugUnits and the targeting moiety can be about 25:1, 20:1, 15:1, 10:1, 5:1or 2:1.

For example, the conjugate disclosed herein is used for the manufactureof a medicament useful for treating or lessening the severity ofdisorders, such as, characterized by abnormal growth of cells (e.g.,cancer).

For example, the Drug Unit or D is locally delivered to a specifictarget cell, tissue, or organ.

The disclosure also provides compositions comprising the conjugates,methods for their preparation, and methods of use thereof in thetreatment of various disorders, including, but not limited to cancer.

In one aspect, the disclosure further relates to a pharmaceuticalcomposition comprising a scaffold or conjugate described herein and apharmaceutically acceptable carrier.

In another aspect, the disclosure relates to a method of treating adisorder in a subject in need thereof, comprising administering to thesubject an effective amount of a conjugate disclosed herein.

In yet another aspect, the disclosure relates to a method of diagnosinga disorder in a subject suspected of having the disorder. The methodcomprises administering an effective amount of the conjugate describedherein to the subject suspected of having the disorder or performing anassay to detect a target antigen/receptor in a sample from the subjectso as to determine whether the subject expresses target antigen orreceptor.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the anti-tumor efficacy of the Trastuzumab-drugconjugate, Conjugate 43B (see Example 18), as measured in a N-87 mousetumor xenograft model.

FIG. 2 illustrates the anti-tumor efficacy of the XMT-1535-drugconjugates: Example 24, Conjugate 53D; Example 25, Conjugate 55A;Example 29, Conjugate 66 as measured in an OVCAR3 mouse tumor xenograftmodel. XMT-1535 is an antibody disclosed in co-pending U.S. applicationSer. No. 15/457,574 filed Mar. 13, 2017.

FIG. 3 illustrates the anti-tumor efficacy of the XMT-1535-drugconjugates: Example 25, Conjugate 55C; Example 26, Conjugate 57; Example28, Conjugate 61; and Example 27, Conjugate 59; as measured in an OVCAR3mouse tumor xenograft model.

FIG. 4 illustrates the anti-tumor efficacy of the Trastuzumab-drugconjugates: Example 30, Conjugate 67; and Example 31, Conjugate 68 asmeasured in a Calu-3 mouse tumor xenograft model.

FIG. 5 illustrates the anti-tumor efficacy of the XMT-1535-drugconjugates: Example 33, Conjugate 76; Example 26, Conjugate 57; Example25, Conjugate 55C; and Example 27, Conjugate 59; as measured in anOVCAR3 mouse tumor xenograft model.

FIG. 6 illustrates the anti-tumor efficacy of the XMT-1535-drugconjugates: Example 26, Conjugate 57; Example 33, Conjugate 76; andExample 34, Conjugate 78; in an OVCAR3 mouse tumor xenograft model.

FIG. 7A, FIG. 7B and FIG. 7C shows the total antibody total AF-HPA andconjugated AF-HPA concentrations respectively for the PBRM-polymer-drugconjugate Example 35, Conjugate 79A; Example 35, Conjugate 7B; Example37, Conjugate 83; and Example 38, Conjugate 85; as measured afteradministration of the conjugates to mice.

DETAILED DESCRIPTION

The present disclosure provides novel targeting moiety-drug conjugates,scaffolds for making the conjugates, synthetic methods for making theconjugates or scaffolds, pharmaceutical compositions containing them andvarious uses of the conjugates.

Certain compounds of the present disclosure and definitions of specificfunctional groups are also described in more detail herein. For purposesof this disclosure, the chemical elements are identified in accordancewith the Periodic Table of the Elements, CAS version, Handbook ofChemistry and Physics, 75^(th) Ed., inside cover, and specificfunctional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in “OrganicChemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999,the entire contents of which are incorporated herein by reference.Furthermore, it will be appreciated by one of ordinary skill in the artthat the synthetic methods, as described herein, utilize a variety ofprotecting groups.

The use of the articles “a”, “an”, and “the” in both the followingdescription and claims are to be construed to cover both the singularand the plural, unless otherwise indicated herein or clearlycontradicted by context. The terms “comprising”, “having”, “being of” asin “being of a chemical formula”, “including”, and “containing” are tobe construed as open terms (i.e., meaning “including but not limitedto”) unless otherwise noted, permits but does not require the inclusionof additional elements or steps. For example, a scaffold of a certainformula includes all components shown in the formula and may alsoinclude additional component not shown in the formula. Additionallywhenever “comprising” or another open-ended term is used in anembodiment, it is to be understood that the same embodiment can be morenarrowly claimed using the intermediate term “consisting essentially of”or the closed term “consisting of.”

As used herein, the expressions “one or more of A, B, or C,” “one ormore A, B, or C,” “one or more of A, B, and C,” “one or more A, B, andC” and the like are used interchangeably and all refer to a selectionfrom a group consisting of A, B, and /or C, i.e., one or more As, one ormore Bs, one or more Cs, or any combination thereof.

The term “about”, “approximately”, or “approximate”, when used inconnection with a numerical value, means that a collection or range ofvalues is included. For example, “about X” includes a range of valuesthat are ±25%, ±20%, ±15%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1%of X, where X is a numerical value. In one embodiment, the term “about”refers to a range of values which are 5% more or less than the specifiedvalue. In another embodiment, the term “about” refers to a range ofvalues which are 2% more or less than the specified value. In anotherembodiment, the term “about” refers to a range of values which are 1%more or less than the specified value.

Recitation of ranges of values are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. A range used herein, unless otherwisespecified, includes the two limits of the range. For example, theexpressions “x being an integer between 1 and 6” and “x being an integerof 1 to 6” both mean “x being 1, 2, 3, 4, 5, or 6”, i.e., the terms“between X and Y” and “range from X to Y, are inclusive of X and Y andthe integers there between.

“Protecting group”: as used herein, the term protecting group means thata particular functional moiety, e.g., O, S, or N, is temporarily blockedso that a reaction can be carried out selectively at another reactivesite in a multifunctional compound. In preferred embodiments, aprotecting group reacts selectively in good yield to give a protectedsubstrate that is stable to the projected reactions; the protectinggroup must be selectively removed in good yield by readily available,preferably nontoxic reagents that do not attack the other functionalgroups; the protecting group forms an easily separable derivative (morepreferably without the generation of new stereogenic centers); and theprotecting group has a minimum of additional functionality to avoidfurther sites of reaction. As detailed herein, oxygen, sulfur, nitrogenand carbon protecting groups may be utilized. For example, in certainembodiments, certain exemplary oxygen protecting groups may be utilized.These oxygen protecting groups include, but are not limited to methylethers, substituted methyl ethers (e.g., MOM (methoxymethyl ether), MTM(methylthiomethyl ether), BOM (benzyloxymethyl ether), and PMBM(p-methoxybenzyloxymethyl ether)), substituted ethyl ethers, substitutedbenzyl ethers, silyl ethers (e.g., TMS (trimethylsilyl ether), TES(triethylsilyl ether), TIPS (triisopropylsilyl ether), TBDMS(t-butyldimethylsilyl ether), tribenzyl silyl ether, and TBDPS(t-butyldiphenyl silyl ether), esters (e.g., formate, acetate, benzoate(Bz), trifluoroacetate, and dichloroacetate), carbonates, cyclic acetalsand ketals. In certain other exemplary embodiments, nitrogen protectinggroups are utilized. Nitrogen protecting groups, as well as protectionand deprotection methods are known in the art. Nitrogen protectinggroups include, but are not limited to, carbamates (including methyl,ethyl and substituted ethyl carbamates (e.g., Troc), amides, cyclicimide derivatives, N-Alkyl and N-Aryl amines, imine derivatives, andenamine derivatives. In yet other embodiments, certain exemplary sulphurprotecting groups may be utilized. The sulfur protecting groups include,but are not limited to those oxygen protecting group describe above aswell as aliphatic carboxylic acid (e.g., acrylic acid), maleimide, vinylsulfonyl, and optionally substituted maleic acid. Certain otherexemplary protecting groups are detailed herein, however, it will beappreciated that the present disclosure is not intended to be limited tothese protecting groups; rather, a variety of additional equivalentprotecting groups can be readily identified using the above criteria andutilized in the present disclosure. Additionally, a variety ofprotecting groups are described in “Protective Groups in OrganicSynthesis” Third Ed. Greene, T. W. and Wuts, P. G., Eds., John Wiley &Sons, New York: 1999, the entire contents of which are herebyincorporated by reference.

“Leaving group” refers to a molecular fragment that departs with a pairof electrons in heterolytic bond cleavage. Leaving groups can be anionsor neutral molecules. Leaving groups include, but are not limited tohalides such as Cl⁻, Br⁻, and I⁻, sulfonate esters, such aspara-toluenesulfonate (“tosylate”, TsO⁻), and RC(O)O— in which R ishydrogen, an aliphatic, heteroaliphatic, carbocyclic, orheterocycloalkyl moiety.

“Antibody” refers to a full-length antibody or functional fragment of anantibody comprising an immunoglobulin. By a “functional fragment” it ismeant a sufficient portion of the immunoglobulin or antibody is providedthat the moiety effectively binds or complexes with the cell surfacemolecule for its target cell population, e.g., human oncofetal antigen.

An immunoglobulin may be purified, generated recombinantly, generatedsynthetically, or combinations thereof, using techniques known to thoseof skill in the art. While immunoglobulins within or derived from IgGantibodies are particularly well-suited for use in the conjugates orscaffolds of this disclosure, immunoglobulins from any of the classes orsubclasses may be selected, e.g., IgG, IgA, IgM, IgD and IgE. Suitably,the immunoglobulin is of the class IgG including but not limited to IgGsubclasses (IgG1, 2, 3 and 4) or class IgM which is able to specificallybind to a specific epitope on an antigen. Antibodies can be intactimmunoglobulins derived from natural sources or from recombinant sourcesand can be immunoreactive portions of intact immunoglobulins. Antibodiesmay exist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, camelized single domain antibodies,intracellular antibodies (“intrabodies”), recombinant antibodies,anti-idiotypic antibodies, domain antibodies, linear antibody,multispecific antibody, antibody fragments, such as, Fv, Fab, F(ab)₂,F(ab)₃, Fab′, Fab′-SH, F(ab′)₂, single chain variable fragmentantibodies (scFv), tandem/bis-scFv, Fc, pFc′, scFvFc (or scFv-Fc),disulfide Fv (dsfv), bispecific antibodies (bc-scFv) such as BiTEantibodies; camelid antibodies, resurfaced antibodies, humanizedantibodies, fully human antibodies, single-domain antibody (sdAb, alsoknown as NANOBODY®), chimeric antibodies, chimeric antibodies comprisingat least one human constant region, dual-affinity antibodies such as,dual-affinity retargeting proteins (DART™), divalent (or bivalent)single-chain variable fragments (di-scFvs, bi-scFvs) including but notlimited to minibodies, diabodies, triabodies or tribodies, tetrabodies,and the like, and multivalent antibodies. “Antibody fragment” refers toat least a portion of the variable region of the immunoglobulin moleculethat binds to its target, i.e., the antigen-binding region. As usedherein, the term “antibody” refers to both the full-length antibody andantibody fragments unless otherwise specified.

“Protein based recognition-molecule” or “PBRM” refers to a molecule thatrecognizes and binds to a cell surface marker or receptor such as, atransmembrane protein, surface immobilized protein, or proteoglycan.Examples of PBRMs include but are not limited to, antibodies (e.g.,Trastuzumab, Cetuximab, Rituximab, Bevacizumab, Epratuzumab, Veltuzumab,Labetuzumab, B7-H4, B7-H3, CA125, CD33, CXCR2, EGFR, FGFR1, FGFR2,FGFR3, FGFR4, HER2, NaPi2b, c-Met, NOTCH1, NOTCH2, NOTCH3, NOTCH4,PD-L1, c-Kit, MUC1, MUC13 and anti-5T4) or peptides (LHRH receptortargeting peptides, EC-1 peptide), lipocalins, such as, for example,anticalins, proteins such as, for example, interferons, lymphokines,growth factors, colony stimulating factors, and the like, peptides orpeptide mimics, and the like. The protein based recognition molecule, inaddition to targeting the conjugate to a specific cell, tissue orlocation, may also have certain therapeutic effect such asantiproliferative (cytostatic and/or cytotoxic) activity against atarget cell or pathway. The protein based recognition molecule comprisesor may be engineered to comprise at least one chemically reactive groupsuch as, —COOH, primary amine, secondary amine —NHR, —SH, or achemically reactive amino acid moiety or side chains such as, forexample, tyrosine, histidine, cysteine, or lysine. In one embodiment, aPBRM may be a ligand (LG) or targeting moiety which specifically bindsor complexes with a cell surface molecule, such as a cell surfacereceptor or antigen, for a given target cell population. Followingspecific binding or complexing of the ligand with its receptor, the cellis permissive for uptake of the ligand or ligand-drug-conjugate, whichis then internalized into the cell. As used herein, a ligand that“specifically binds or complexes with” or “targets” a cell surfacemolecule preferentially associates with a cell surface molecule viaintermolecular forces. For example, the ligand can preferentiallyassociate with the cell surface molecule with a Kd of less than about 50nM, less than about 5 nM, or less than 500 pM. Techniques for measuringbinding affinity of a ligand to a cell surface molecule are well-known;for example, one suitable technique, is termed surface plasmon resonance(SPR). In one embodiment, the ligand is used for targeting and has nodetectable therapeutic effect as separate from the drug which itdelivers. In another embodiment, the ligand functions both as atargeting moiety and as a therapeutic or immunomodulatory agent (e.g.,to enhance the activity of the active drug or prodrug).

“Biocompatible” as used herein is intended to describe compounds thatexert minimal destructive or host response effects while in contact withbody fluids or living cells or tissues. Thus a biocompatible group, asused herein, refers to an aliphatic, cycloalkyl, heteroaliphatic,heterocycloalkyl, aryl, or heteroaryl moiety, which falls within thedefinition of the term biocompatible, as defined above and herein. Theterm “Biocompatibility” as used herein, is also taken to mean that thecompounds exhibit minimal interactions with recognition proteins, e.g.,naturally occurring antibodies, cell proteins, cells and othercomponents of biological systems, unless such interactions arespecifically desirable. Thus, substances and functional groupsspecifically intended to cause the above minimal interactions, e.g.,drugs and prodrugs, are considered to be biocompatible. Preferably (withexception of compounds intended to be cytotoxic, such as, e.g.,antineoplastic agents), compounds are “biocompatible” if their additionto normal cells in vitro, at concentrations similar to the intendedsystemic in vivo concentrations, results in less than or equal to 1%cell death during the time equivalent to the half-life of the compoundin vivo (e.g., the period of time required for 50% of the compoundadministered in vivo to be eliminated/cleared), and their administrationin vivo induces minimal and medically acceptable inflammation, foreignbody reaction, immunotoxicity, chemical toxicity and/or other suchadverse effects. In the above sentence, the term “normal cells” refersto cells that are not intended to be destroyed or otherwisesignificantly affected by the compound being tested.

“Biodegradable”: As used herein, “biodegradable” compounds or moietiesare those that, when taken up by cells, can be broken down by thelysosomal or other chemical machinery or by hydrolysis into componentsthat the cells can either reuse or dispose of without significant toxiceffect on the cells. The term “biocleavable” as used herein has the samemeaning of “biodegradable”. The degradation fragments preferably inducelittle or no organ or cell overload or pathological processes caused bysuch overload or other adverse effects in vivo. Examples ofbiodegradation processes include enzymatic and non-enzymatic hydrolysis,oxidation and reduction. Suitable conditions for non-enzymatichydrolysis of the biodegradable conjugates (or their components, e.g.,the peptide-containing scaffolds and the linkers between the scaffoldsand the antibody or the drug molecule) described herein, for example,include exposure of the biodegradable conjugates to water at atemperature and a pH of lysosomal intracellular compartment.Biodegradation of some conjugates (or their components, e.g., thepeptide-containing scaffolds and the linkers between the scaffolds andthe antibody or the drug molecule), can also be enhancedextracellularly, e.g., in low pH regions of the animal body, e.g., aninflamed area, in the close vicinity of activated macrophages or othercells releasing degradation facilitating factors. The integrity of theconjugates or scaffolds disclosed herein can be measured, for example,by size exclusion HPLC. Although faster degradation may be in some casespreferable, in general it may be more desirable that the conjugates orscaffolds disclosed herein degrade in cells with the rate that does notexceed the rate of metabolization or excretion of their fragments by thecells. In preferred embodiments, the biodegradation byproducts ofconjugates or scaffolds disclosed herein are biocompatible.

“Bioavailability”: The term “bioavailability” refers to the systemicavailability (i.e., blood/plasma levels) of a given amount of drug orcompound administered to a subject. Bioavailability is an absolute termthat indicates measurement of both the time (rate) and total amount(extent) of drug or compound that reaches the general circulation froman administered dosage form.

“Hydrophilic”: The term “hydrophilic” does not essentially differ fromthe common meaning of this term in the art, and denotes chemicalmoieties which contain ionizable, polar, or polarizable atoms, or whichotherwise may be solvated by water molecules. Thus a hydrophilic moietyor group, as used herein, refers to an aliphatic, cycloalkyl,heteroaliphatic, heterocycloalkyl, aryl or heteroaryl moiety, whichfalls within the definition of the term hydrophilic, as defined above.Examples of particular hydrophilic organic moieties which are suitableinclude, without limitation, aliphatic or heteroaliphatic groupscomprising a chain of atoms in a range of between about one and twelveatoms, hydroxyl, hydroxyalkyl, amine, carboxyl, amide, carboxylic ester,thioester, aldehyde, nitryl, isonitryl, nitroso, hydroxylamine,mercaptoalkyl, heterocycle, carbamates, carboxylic acids and theirsalts, sulfonic acids and their salts, sulfonic acid esters, phosphoricacids and their salts, phosphate esters, polyglycol ethers, polyamines,polycarboxylates, polyesters, polythioesters, polyalcohols andderivatives thereof. In certain embodiments, hydrophilic substituentscomprise a carboxyl group (COOH), an aldehyde group (CHO), a ketonegroup (COC₁₋₄ alkyl), a methylol (CH₂OH) or a glycol (for example,CHOH—CH₂OH or CH—(CH2OH)₂), NH₂, F, cyano, SO₃H, PO₃H, and the like.

Hydrophilicity of the compounds (including drugs, conjugates andscaffolds) disclosed herein can be directly measured throughdetermination of hydration energy, or determined through investigationbetween two liquid phases, or by chromatography on solid phases withknown hydrophobicity, such as, for example, C4 or C18.

“Physiological conditions”: The phrase “physiological conditions”, asused herein, relates to the range of chemical (e.g., pH, ionic strength)and biochemical (e.g., enzyme concentrations) conditions likely to beencountered in the extracellular fluids of living tissues. For mostnormal tissues, the physiological pH ranges from about 7.0 to 7.4.Circulating blood plasma and normal interstitial liquid representtypical examples of normal physiological conditions.

“Polysaccharide”, “carbohydrate” or “oligosaccharide”: The terms“polysaccharide”, “carbohydrate”, or “oligosaccharide” are known in theart and refer, generally, to substances having chemical formula(CH₂O)_(n), where generally n>2, and their derivatives. Carbohydratesare polyhydroxyaldehydes or polyhydroxyketones, or change to suchsubstances on simple chemical transformations, such as hydrolysis,oxidation or reduction. Typically, carbohydrates are present in the formof cyclic acetals or ketals (such as, glucose or fructose). These cyclicunits (monosaccharides) may be connected to each other to form moleculeswith few (oligosaccharides) or several (polysaccharides) monosaccharideunits. Often, carbohydrates with well defined number, types andpositioning of monosaccharide units are called oligosaccharides, whereascarbohydrates consisting of mixtures of molecules of variable numbersand/or positioning of monosaccharide units are called polysaccharides.The terms “polysaccharide”, “carbohydrate”, and “oligosaccharide”, areused herein interchangeably. A polysaccharide may include natural sugars(e.g., glucose, fructose, galactose, mannose, arabinose, ribose, andxylose) and/or derivatives of naturally occurring sugars (e.g.,2′-fluororibose, 2′-deoxyribose, and hexose).

“Drug”: As used herein, the term “drug” refers to a compound which isbiologically active and provides a desired physiological effectfollowing administration to a subject in need thereof (e.g., an activepharmaceutical ingredient).

“Prodrug”: As used herein the term “prodrug” refers to a precursor of anactive drug, that is, a compound that can be transformed to an activedrug. Typically such a prodrug is subject to processing in vivo, whichconverts the drug to a physiologically active form. In some instances, aprodrug may itself have a desired physiologic effect. A desiredphysiologic effect may be, e.g., therapeutic, cytotoxic,immunomodulatory, or the like.

“Cytotoxic”: As used herein the term “cytotoxic” means toxic to cells ora selected cell population (e.g., cancer cells). The toxic effect mayresult in cell death and/or lysis. In certain instances, the toxiceffect may be a sublethal destructive effect on the cell, e.g., slowingor arresting cell growth. In order to achieve a cytotoxic effect, thedrug or prodrug may be selected from a group consisting of a DNAdamaging agent, a microtubule disrupting agent, or a cytotoxic proteinor polypeptide, amongst others.

“Cytostatic”: As used herein the term “cytostatic” refers to a drug orother compound which inhibits or stops cell growth and/ormultiplication.

“Small molecule”: As used herein, the term “small molecule” refers tomolecules, whether naturally-occurring or artificially created (e.g.,via chemical synthesis) that have a relatively low molecular weight.Preferred small molecules are biologically active in that they produce alocal or systemic effect in animals, preferably mammals, more preferablyhumans. In certain preferred embodiments, the small molecule is a drugand the small molecule is referred to as “drug molecule” or “drug” or“therapeutic agent”. In certain embodiments, the drug molecule has MWless than or equal to about 5 kDa. In other embodiments, the drugmolecule has MW less than or equal to about 1.5 kDa. In embodiments, thedrug molecule is selected from vinca alkaloids, auristatins,duocarmycins, kinase inhibitors, MEK inhibitors, KSP inhibitors, PI3kinase inhibitors, calicheamicins, SN38, camptothecin, topoisomeraseinhibitors, non-natural camptothecins, protein synthesis inhibitor, RNApolymerase inhibitor, pyrrolobenzodiazepines, maytansinoids, DNA-bindingdrugs, DNA intercalation drugs and analogs thereof. Preferably, thoughnot necessarily, the drug is one that has already been deemed safe andeffective for use by an appropriate governmental agency or body, e.g.,the FDA. For example, drugs for human use listed by the FDA under 21C.F.R. §§ 330.5, 331 through 361, and 440 through 460; drugs forveterinary use listed by the FDA under 21 C.F.R. §§ 500 through 589,incorporated herein by reference, are all considered suitable for themethods, conjugates, and scaffolds disclosed herein. Classes of drugmolecules that can be used in the practice of the present inventioninclude, but are not limited to, anti-cancer substances, radionuclides,vitamins, anti-AIDS substances, antibiotics, immunosuppressants,anti-viral substances, enzyme inhibitors, neurotoxins, opioids,hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants,muscle relaxants and anti-Parkinson substances, anti-spasmodics andmuscle contractants including channel blockers, miotics andanti-cholinergics, anti-glaucoma compounds, anti-parasite and/oranti-protozoal compounds, modulators of cell-extracellular matrixinteractions including cell growth inhibitors and anti-adhesionmolecules, vasodilating agents, inhibitors of DNA, RNA or proteinsynthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal andnon-steroidal anti-inflammatory agents, anti-angiogenic factors,anti-secretory factors, anticoagulants and/or antithrombotic agents,local anesthetics, ophthalmics, prostaglandins, anti-depressants,anti-psychotic substances, anti-emetics, imaging agents. Many largemolecules are also drugs and such large molecules may be used in theconjugates and other constructs described herein. Examples of suitablelarge molecules include, e.g., amino acid based molecules. Amino acidbased molecules may encompass, e.g., peptides, polypeptides, enzymes,antibodies, immunoglobulins, or functional fragments thereof, amongothers.

A more complete, although not exhaustive, listing of classes andspecific drugs suitable for use in the present disclosure may be foundin “Pharmaceutical Substances: Syntheses, Patents, Applications” by AxelKleemann and Jurgen Engel, Thieme Medical Publishing, 1999 and the“Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals”,Edited by Susan Budavari et al., CRC Press, 1996, both of which areincorporated herein by reference. In preferred embodiments, the drugused in this disclosure is a therapeutic agent that hasantiproliferative (cytostatic and/or cytotoxic) activity against atarget cell or pathway. The drug may have a chemically reactive groupsuch as, for example, —COOH, primary amine, secondary amine —NHR, —OH,—SH, —C(O)H, —C(O)R, —C(O)NHR^(2b), C(S)OH, —S(O)₂OR^(2b),—P(O)₂OR^(2b), —CN, —NC or —ONO, in which R is an aliphatic,heteroaliphatic, carbocyclic or heterocycloalkyl moiety and R^(2b) is ahydrogen, an aliphatic, heteroaliphatic, carbocyclic, or heterocyclicmoiety.

“Active form” as used herein refers to a form of a compound thatexhibits intended pharmaceutical efficacy in vivo or in vitro. Inparticular, when a drug molecule intended to be delivered by theconjugate of the disclosure is released from the conjugate, the activeform can be the drug itself or its derivatives, which exhibit theintended therapeutic properties. The release of the drug from theconjugate can be achieved by cleavage of a biodegradable bond of thelinker which attaches the drug to the scaffold or conjugate of thedisclosure. The active drug derivatives accordingly can comprise aportion of the linker.

“Diagnostic label”: As used herein, the term diagnostic label refers toan atom, group of atoms, moiety or functional group, a nanocrystal, orother discrete element of a composition of matter, that can be detectedin vivo or ex vivo using analytical methods known in the art. Whenassociated with a conjugate of the present disclosure, such diagnosticlabels permit the monitoring of the conjugate in vivo. Alternatively oradditionally, constructs and compositions that include diagnostic labelscan be used to monitor biological functions or structures. Examples ofdiagnostic labels include, without limitation, labels that can be usedin medical diagnostic procedures, such as, radioactive isotopes(radionuclides) for gamma scintigraphy and Positron Emission Tomography(PET), contrast agents for Magnetic Resonance Imaging (MM) (for exampleparamagnetic atoms and superparamagnetic nanocrystals), contrast agentsfor computed tomography and other X-ray-based imaging methods, agentsfor ultrasound-based diagnostic methods (sonography), agents for neutronactivation (e.g., boron, gadolinium), fluorophores for various opticalprocedures, and, in general moieties which can emit, reflect, absorb,scatter or otherwise affect electromagnetic fields or waves (e.g.,gamma-rays, X-rays, radiowaves, microwaves, light), particles (e.g.,alpha particles, electrons, positrons, neutrons, protons) or other formsof radiation, e.g., ultrasound.

“Animal”: The term animal, as used herein, refers to humans as well asnon-human animals, at any stage of development, including, for example,mammals, birds, reptiles, amphibians, fish, worms and single cells. Cellcultures and live tissue samples are considered to be pluralities ofanimals. Preferably, the non-human animal is a mammal (e.g., a rodent, amouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). Ananimal may be a transgenic animal or a human clone. The term “subject”encompasses animals.

“Efficient amount”: In general, as it refers to an active agent or drugdelivery device, the term “efficient amount” refers to the amountnecessary to elicit the desired biological response. As will beappreciated by those of ordinary skill in this art, the efficient amountof an agent or device may vary depending on such factors as the desiredbiological endpoint, the agent to be delivered, the composition of theencapsulating matrix, the target tissue, etc. For example, the efficientamount of microparticles containing an antigen to be delivered toimmunize an individual is the amount that results in an immune responsesufficient to prevent infection with an organism having the administeredantigen.

“Natural amino acid” as used herein refers to any one of the common,naturally occurring L-amino acids found in naturally occurring proteins,such as, glycine (Gly), alanine (Ala), valine (Val), leucine (Leu),isoleucine (Ile), lysine (Lys), arginine (Arg), histidine (His), proline(Pro), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine(Tyr), tryptophan (Trp), aspartic acid (Asp), glutamic acid (Glu),asparagine (Asn), glutamine (Gln), cysteine (Cys), methionine (Met) or astereoisomer thereof, e.g., isoglutamic acid (iGlu) or isoaspartic acid(iAsp). Unless specified otherwise, a reference to an amino acidincludes the amino acid itself and its stereoisomers. For example, theterm “glutamic acid” includes both Glu and iGlu while the term “asparticacid” includes both Asp and iAsp.

“Unnatural amino acid” as used herein refers to any amino acid which isnot a natural amino acid. This includes, for example, amino acids thatcomprise α-, β-, γ-, D-, L-amino acyl residues. More generally, theunnatural amino acid comprises a residue of the general formula

wherein the side chain R is other than the amino acid side chainsoccurring in nature. Exemplary unnatural amino acids, include, but arenot limited to, sarcosine (N-methylglycine), citrulline (cit),homocitrulline, β-ureidoalanine, thiocitrulline, hydroxyproline,allothreonine, pipecolic acid (homoproline), α-aminoisobutyric acid,tert-butylglycine, tert-butylalanine, allo-isoleucine, norleucine,α-methylleucine, cyclohexylglycine, β-cyclohexylalanine,β-cyclopentylalanine, α-methylproline, phenylglycine,α-methylphenylalanine and homophenylalanine.

“Alkyl” by itself or as part of another term, as used herein, refers toa substituted or unsubstituted straight chain or branched, saturated orunsaturated hydrocarbon having the indicated number of carbon atoms(e.g., “—C₁₋₈ alkyl” or “—C₁₋₁₀ alkyl refer to an alkyl group havingfrom 1 to 8 or 1 to 10 carbon atoms, respectively). When the number ofcarbon atoms is not indicated, the alkyl group has from 1 to 8 carbonatoms. Representative straight chain “—C₁₋₈ alkyl” groups include, butare not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl,-n-hexyl, -n-heptyl and -n-octyl; while branched —C₁₋₈ alkyls include,but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl,-isopentyl, and -2-methylbutyl; unsaturated —C₂₋₈ alkyls include, butare not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl,-isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl,-2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, -1-hexyl, 2-hexyl,-3-hexyl, -acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl,-2-pentynyl and -3-methyl-1 butynyl. In some embodiments, an alkyl groupis unsubstituted. An alkyl group can be substituted with one or moregroups. In other aspects, an alkyl group will be saturated.

“Alkylene” by itself of as part of another term, as used herein, refersto a substituted or unsubstituted saturated or unsaturated branched orstraight chain or cyclic hydrocarbon radical of the stated number ofcarbon atoms, typically 1-10 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—), 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like. In some embodiments, an alkylene is a branched or straightchain hydrocarbon (i.e., it is not a cyclic hydrocarbon). In any of theembodiments provided herein, the alkylene can be a saturated alkylene.

“Aryl” by itself or as part of another term, as used herein, means asubstituted or unsubstituted monovalent carbocyclic aromatic hydrocarbonradical of 6-20 carbon (preferably 6-14 carbon) atoms derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Some aryl groups are represented in the exemplarystructures as “Ar”. Typical aryl groups include, but are not limited to,radicals derived from benzene, substituted benzene, naphthalene,anthracene, biphenyl, and the like. An exemplary aryl group is a phenylgroup.

“Arylene” by itself or as part of another term, as used herein, is anaryl group as defined above wherein one of the aryl group's hydrogenatoms is replaced with a bond (i.e., it is divalent) and can be in theortho, meta, or para orientations as shown in the following structures,with phenyl as the exemplary group:

In some embodiments, e.g., when a Multifunctional Linker or Drug Unit,comprises an arylene, the arylene is an aryl group defined above whereinone or two of the aryl group's hydrogen atoms is replaced with a bond(i.e., the arylene can be divalent or trivalent).

“Heterocycle” by itself or as part of another term, as used herein,refers to a monovalent substituted or unsubstituted aromatic(“heteroaryl”) or non-aromatic (“heterocycloalkyl”) monocyclic,bicyclic, tricyclic, or tetracyclic ring system having a certain numberof (e.g., from 3 to 8 or C₃₋₈) carbon atoms (also referred to as ringmembers) and one to four heteroatom ring members independently selectedfrom N, O, P or S, and derived by removal of one hydrogen atom from aring atom of a parent ring system. One or more N, C or S atoms in theheterocycle can be oxidized. The ring that includes the heteroatom canbe aromatic or nonaromatic. Unless otherwise noted, the heterocycle isattached to its pendant group at any heteroatom or carbon atom thatresults in a stable structure. Representative examples of a heterocycle(e.g., C₃₋₈ heterocycle) include, but are not limited to, pyrrolidinyl,azetidinyl, piperidinyl, morpholinyl, tetrahydrofuranyl,tetrahydropyranyl, benzofuranyl, benzothiophene, indolyl,benzopyrazolyl, pyrrolyl, thiophenyl (thiophene), furanyl, thiazolyl,imidazolyl, pyrazolyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl,isothiazolyl, and isoxazolyl.

“Heterocyclo” or “Heterocyclo-” when used herein, refers to aheterocycle group (e.g., C₃₋₈ heterocycle) defined above wherein one ormore of additional hydrogen atoms of the heterocycle are replaced with abond (i.e., it is multivalent, such as divalent or trivalent). In someembodiments, when a hydrophilic group, Multifunctional Linker orLinker-Drug moiety comprises a heterocyclo, the heterocyclo is aheterocycle group defined above wherein one or two of the heterocyclegroup's hydrogen atoms is replaced with a bond (i.e., the heterocyclocan be divalent or trivalent).

“Carbocycle” by itself or as part of another term, when used herein, ismonovalent, substituted or unsubstituted, aromatic (“aryl”) or saturatedor unsaturated non-aromatic (“cycloalkyl”), monocyclic, bicyclic,tricyclic, or tetracyclic carbocyclic ring system having a certainnumber of (e.g., from 3 to 8 or C₃₋₈) carbon atoms (also referred to asring members) derived by the removal of one hydrogen atom from a ringatom of a parent ring system. A carbocycle can be 3-, 4-, 5-, 6-, 7- or8-membered. Representative C₃₋₈ carbocycles include, but are not limitedto, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl,cyclohexenyl, phenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl,cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctyl,and cyclooctadienyl.

“Carbocyclo” or “Carbocyclo-” by itself or as part of another term, whenused herein, refers to a C₃₋₈ carbocycle group defined above whereinanother of the carbocycle groups' hydrogen atoms is replaced with a bond(i.e., it is divalent). In select embodiments, e.g., when a hydrophilicgroup, Multifunctional Linker or Linker-Drug moiety comprises acarbocyclo, the carbocyclo is a carbocycle group defined above whereinone or two of the carbocycle group's hydrogen atoms is replaced with abond (i.e., the carbocyclo can be divalent or trivalent).

“Heteroalkyl” by itself or in combination with another term, when usedherein, means, unless otherwise stated, a stable straight or branchedchain hydrocarbon, or combinations thereof, fully saturated orcontaining from 1 to 3 degrees of unsaturation, consisting of the statednumber of carbon atoms and from one to ten, preferably one to three,heteroatoms selected from the group consisting of O, N, Si and S, andwherein the nitrogen and sulfur atoms may optionally be oxidized and thenitrogen heteroatom may optionally be quaternized. The heteroatom(s) O,N and S may be placed at any interior position of the heteroalkyl groupor at the position at which the alkyl group is attached to the remainderof the molecule. The heteroatom Si may be placed at any position of theheteroalkyl group, including the position at which the alkyl group isattached to the remainder of the molecule. Examples include—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂—S(O)—CH₃, —NH—CH₂—CH₂—NH—C(O)—CH₂—CH₃, —CH₂—CH₂—S(O)₂—CH₃,—CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—O—CH₃, and —CH═CH—N(CH₃)—CH₃. Up totwo heteroatoms may be consecutive, such as, for example, —CH₂—NH—OCH₃and —CH₂—O—Si(CH₃)₃. In preferred embodiments, a C₁₋₄ heteroalkyl orheteroalkylene has 1 to 4 carbon atoms and 1 or 2 heteroatoms and a C₁₋₃heteroalkyl or heteroalkylene has 1 to 3 carbon atoms and 1 or 2heteroatoms. In some aspects, a heteroalkyl or heteroalkylene issaturated.

“Heteroalkylene” by itself or as part of another substituent, when usedherein, means a divalent group derived from heteroalkyl (as discussedabove), as exemplified by —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini. Still further, for alkyleneand heteroalkylene linking groups, no orientation of the linking groupis implied. In select embodiments, e.g., when a hydrophilic group,Multifunctional Linker or Linker-Drug moiety comprises a heteroalkylene,the heteroalkylene is a heteroalkyl group defined above wherein one ortwo of the heteroalkyl group's hydrogen atoms is replaced with a bond(i.e., the heteroalkylene can be divalent or trivalent).

“Optionally substituted” when used herein, means that a chemical moiety(such as alkyl, heteroalkyl, carbocycle, and heterocycle, etc.) iseither substituted or unsubstituted. Unless otherwise specified, thechemical moieties disclosed herein are optionally substituted. When achemical moiety is substituted, one or more hydrogen atoms are eachindependently replaced with a substituent. Typical substituents include,but are not limited to, —X′, —R′, —O, —OR′, —SR′, —S⁻, —N(R′)₂, —N(R′)₃,═NR′, —C(X′)₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃,—NR′C(═O)R′, —C(═O)R′, —C(═O)N(R′)₂, —SO₃ ⁻, —SO₃H, —S(═O)₂R′,—OS(═O)₂OR′, —S(═O)₂NR′, —S(═O)R′, —OP(═O)(OR′)₂, —P(═O)(OR′)₂, —PO₃ ⁻,—PO₃H₂, —AsO₂H₂, —C(═O)R′, —C(═O)X′, —C(═S)R′, —CO₂R′, —CO₂ ⁻,—C(═S)OR′, C(═O)SR′, C(═S)SR′, C(═O)N(R′)₂, C(═S)N(R′)₂, orC(═NR′)N(R′)₂, wherein each X′ is independently a halogen: —F, —Cl, —Br,or —I; and each R′ is independently —H, —C₁₋₂₀ alkyl, —C₆₋₂₀ aryl,—C₃-C₁₄ heterocycle, a protecting group or a prodrug moiety. Typicalsubstituents also include oxo (═O).

“Linker-Drug moiety” as used herein, refers to the non-targeting moietyportion of a conjugate disclosed herein. The Linker component of theLinker-Drug moiety has the release mechanism, which is referred to asthe Releasable Assembly Unit, interposed between a MultifunctionalLinker and a Drug Unit.

“Multifunctional Linker” as used herein, refers to a linker thatconnects one or more hydrophilic groups, one or more Drug Units, and atargeting moiety (e.g., a PBRM) to form a conjugate or scaffold asdisclosed herein. The connection of these components to theMultifunctional Linker can either be parallel or serial. In someembodiments, the Multifunctional Linker comprises a peptide moietybetween the targeting moiety and the hydrophilic group, wherein thepeptide moiety includes at least two amino acids. In other embodiments,the Multifunctional Linker does not have to comprise a peptide moiety ofat least two amino acids when the hydrophilic group is a polyalcohol ora derivative thereof. In other embodiments, the Multifunctional Linkerdoes not have to comprise a peptide moiety of at least two amino acidswhen the hydrophilic group is a glucosyl-amine, a di-glucosyl-amine, atri-glucosyl-amine or a derivative thereof.

As used herein, the phrase “parallel orientation”, “parallel placement”,“parallel connection” or like terms refer to a configuration wherein theparallel-placed or parallel-oriented or parallel-connected componentsare attached to the Multifunctional Linker in such a manner that eachhas one end tethered to the Multifunctional Linker and one free end. Theterm “parallel” is used herein is not being used to denote that twocomponents are side-by-side in space or have the same distance betweenthem throughout some or their entire lengths. In instances where aparallel-oriented component is itself branched and thus has multipleends, it still has only one tethered end. In some embodiments, onlythose hydrophilic groups, required to mask hydrophobicity for a givenLinker-Drug moiety are in parallel orientation to the Drug Unit, whichdoes not necessarily require all of the Drug Units and hydrophilicgroups connected to the Multifunctional Linker be in parallelorientations to one another. In other embodiments, all of the Drug Unitsand hydrophilic groups connected to the Multifunctional Linker are inparallel orientations to one another.

The phrase “serial orientation” or “serial placement” or “serialconnection” or like terms refer to a configuration of a component in aconjugate or scaffold of the disclosure wherein the serially-orientedcomponent is attached in such a manner that it has two tethered endswith each end connected to a different component of the conjugate orscaffold of the disclosure. For example, one or more (OCH₂CH₂) subunits,which characterize a PEG unit or subunit, are interposed between theDrug Unit and the targeting moiety.

“Free drug” as used herein, refers to a biologically active form of adrug moiety that is not covalently attached either directly orindirectly to a hydrophilic group or to a degradant product of a LigandUnit. Free drug can refer to the drug, as it exists immediately uponcleavage from the Multifunctional Linker via the release mechanism,which is provided by the Releasable Assembly Unit in the Linker-Drugmoiety, or, to subsequent intracellular conversion or metabolism. Insome aspects, the free drug will have the form H-D or may exist a as acharged moiety. The free drug is a pharmacologically active specieswhich can exert the desired biological effect. In some aspects, thepharmacologically active species may not be the parent drug and mayinclude a component of the linker through which the drug is connected tothe targeting moiety, which has not undergone subsequent intracellularmetabolism.

Hydrophobicity can be measured using SlogP. SlogP is defined as the logof the octanol/water partition coefficient (including implicithydrogens) and can be calculated using the program MOE™ from theChemical Computing group (SlogP values calculated using Wildman, S. A.,Crippen, G. M.; Prediction of Physiochemical Parameters by AtomicContributions; J. Chem. Inf. Comput. Sci. 39 No. 5 (1999) 868-873).

In certain embodiments, the present disclosure provides a targetingmoiety-drug conjugate composition comprising a population of targetingmoiety-drug conjugates. The targeting moiety-drug conjugate comprises atargeting moiety unit and multiple Linker-Drug moieties attachedthereto. Preferably, there is an average of from about 2 to about 14,about 3 to about 10, or about 3 to about 5 Linker-Drug moieties (e.g.,d₁₃ of Formula (I)) per targeting moiety in the conjugate. Exemplaryattachment to the targeting moiety is via thioether linkages. Exemplaryconjugation sites on a targeting moiety are the thiol groups obtainedfrom reduction of interchain disulfide residues and/or thiol-containingresidues introduced into the targeting moiety such as introducedcysteines. Attachment can be, for example, via thiol residues derivedfrom an interchain disulfide and from 0 to 8 introduced cysteineresidues.

As used herein, “molecular weight” or “MW” of a polymer refers to theweight average molecular weight unless otherwise specified.

The present disclosure is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium. Isotopes of carbon include C-13 and C-14.

The present disclosure is intended to include all isomers of thecompound (e.g., the drug, conjugate, and scaffold disclosed herein),which refers to and includes, optical isomers, and tautomeric isomers,where optical isomers include enantiomers and diastereomers, chiralisomers and non-chiral isomers, and the optical isomers include isolatedoptical isomers as well as mixtures of optical isomers including racemicand non-racemic mixtures; where an isomer may be in isolated form or ina mixture with one or more other isomers.

Conjugates and Peptide-Containing Scaffolds

In one aspect, the disclosure relates to a conjugate of Formula (I) witha protein based recognition-molecule (PBRM):

wherein

a₁ is an integer from 0 to 1;

a₂ is an integer from 1 to 3;

a₃ is an integer from 0 to 1;

a₄ is an integer from 1 to about 5;

a₅ is an integer from 1 to 3;

d₁₃ is an integer from 1 to about 14;

PBRM denotes a protein based recognition-molecule;

L^(P′) is a divalent linker moiety connecting the PBRM to M^(P); ofwhich the corresponding monovalent moiety L^(P) contains a functionalgroup W^(P) that is capable of forming a covalent bond with a functionalgroup of the PBRM;

M^(P) is a Stretcher unit;

L^(M) is a bond, or a trivalent or tetravalent linker, and when L^(M) isa bond, a₂ is 1, when L^(M) is trivalent linker, a₂ is 2, or when L^(M)is a tetravalent linker, a₂ is 3;

L³ is a carbonyl-containing moiety;

M^(A) comprises a peptide moiety that contains at least two amino acids;

T¹ is a hydrophilic group and the

between T¹ and M^(A) denotes direct or indirect attachment of T¹ andM^(A);

each occurrence of D is independently a therapeutic agent having amolecular weight≤about 5 kDa; and

each occurrence of L′ is independently a divalent linker moietyconnecting D to M^(A) and comprises at least one cleavable bond suchthat when the bond is broken, D is released in an active form for itsintended therapeutic effect.

In another aspect, the disclosure relates to a peptide-containingscaffold of any one of Formulae (II)-(XIV):

wherein

a₁ is an integer from 0 to 1;

a₂ is an integer from 1 to 3;

a₃ is an integer from 0 to 1;

a₄ is an integer from 1 to about 5;

a₅ is an integer from 1 to 3;

d₁₃ is an integer from 1 to about 10;

PBRM denotes a protein based recognition-molecule;

L^(P′) is a divalent linker moiety connecting the PBRM to M^(P); ofwhich the corresponding monovalent moiety L^(P) contains a functionalgroup W^(P) that is capable of forming a covalent bond with a functionalgroup of the PBRM;

M^(P) is a Stretcher unit;

L^(M) is a bond, or a trivalent or tetravalent linker, and when L^(M) isa bond, a₂ is 1, when L^(M) is trivalent linker, a₂ is 2, or when L^(M)is a tetravalent linker, a₂ is 3;

L³ is a carbonyl-containing moiety;

M^(A) comprises a peptide moiety that contains at least two amino acids;

T¹ is a hydrophilic group and the

between T¹ and M^(A) denotes direct or indirect attachment of T¹ andM^(A);

each occurrence of W^(M) is independently hydrogen, a protecting group,a leaving group, or a functional group that is capable of connectingL^(M) to M^(P) by forming a covalent bond;

each occurrence of W^(D) is independently a functional group that iscapable of forming a covalent bond with a functional group of atherapeutic agent (“D”) having a molecular weight≤about 5 kDa; and

each occurrence of L^(D) is independently a divalent linker moietyconnecting W^(D) or D to M^(A) and L^(D) comprises at least onecleavable bond such that when the bond is broken, D is released in anactive form for its intended therapeutic effect.

The conjugates and scaffolds of the disclosure can include one or moreof the following features when applicable.

In one embodiment, d₁₃ is an integer from 2 to 14, from 2 to 12, from 2to 10, from 2 to 8, from 2 to 6, from 2 to 4, from 4 to 10, from 4 to 8,from 4 to 6, from 6 to 14, from 6 to 12, from 6 to 10, from 6 to 8, from8 to 14, from 8 to 12, or from 8 to 10.

In one embodiment, d₁₃ is an integer from 2 to 4 (e.g., d₁₃ is 2, 3, or4).

In one embodiment, d₁₃ is an integer from 4 to 6 (e.g., d₁₃ is 4, 5, or6).

In one embodiment, d₁₃ is an integer from 6 to 8 (e.g., d₁₃ is 6, 7, or8).

In one embodiment, d₁₃ is an integer from 6 to 10 (e.g., d₁₃ is 6, 7, 8,9, or 10).

In a specific embodiment, d₁₃ is 4 or 5.

In some embodiments, L³, when present, comprises —X—C₁₋₁₀alkylene-C(O)—, with X directly connected to L^(M), in which X is CH₂,O, or NR₅, and R₅ is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈ cycloalkyl,COOH, or COO—C₁₋₆ alkyl.

In some embodiments, L³ is —NR₅—(CH₂)_(v)—C(O)— or—CH₂—(CH₂)_(v)—C(O)—NR₅—(CH₂)_(v)—C(O)—, in which each v independentlyis an integer from 1 to 10. For example, L³, when present, is—NH—(CH₂)₂—C(O)— or —(CH₂)₂—C(O)—NH—(CH₂)₂—C(O)—.

In one embodiment, each v independently is an integer from 1 to 6, orfrom 2 to 4, or v is 2.

In one embodiment, a₄ is 1.

In one embodiment, a₄ is 2.

In one embodiment, a₄ is 3.

L^(P) and L^(P′)

L^(P), when not connected to PBRM, comprises a terminal group W^(P), inwhich each W^(P) independently is:

wherein

ring A is cycloalkyl or heterocycloalkyl;

ring B is cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;

R^(1K) is a leaving group;

R^(1A) is a sulfur protecting group;

R^(1J) is hydrogen, an aliphatic, heteroaliphatic, carbocyclic, orheterocycloalkyl moiety;

R^(2J) is hydrogen, an aliphatic, aryl, heteroaliphatic, or carbocyclicmoiety;

R^(3J) is C₁₋₆ alkyl and each of Z₁, Z₂, Z₃ and Z₇ is independently acarbon or nitrogen atom;

R^(4j) is hydrogen, halogen, OR, —NO₂, —CN, —S(O)₂R, C₁₋₂₄ alkyl (e.g.,C₁₋₆ alkyl), or 6-24 membered aryl or heteroaryl, wherein the C₁₋₂₄alkyl (e.g., C₁₋₆ alkyl), or 6-24 membered aryl or heteroaryl, isoptionally substituted with one or more aryl or heteroaryl; or twoR^(4j) together form an annelated cycloalkyl, heterocycloalkyl, aryl, orheteroaryl; R is hydrogen, alkyl, heteroalkyl, cycloalkyl, orheterocycloalkyl;

R^(5j) is C(R^(4j))₂, O, S or NR; and

z₁ is an integer 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

L^(P′) is a divalent linker moiety connecting the PBRM to M^(P); ofwhich the corresponding monovalent moiety is L^(P).

For example, each R^(1K) is halo or RC(O)O— in which R is hydrogen, analiphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

For example, each R^(1A) independently is

in which r is 1 or 2 and each of R^(s1), R^(s2), and R^(s3) is hydrogen,an aliphatic, heteroaliphatic, carbocyclic, or heterocycloalkyl moiety.

For example, ring A can be C₃₋₈ cycloalkyl or 5-19 memberedheterocycloalkyl.

For example, ring A can be

wherein R^(6j) is hydrogen, halogen, C₁₋₂₄ alkyl (e.g., C₁₋₆ alkyl), or6-24 membered aryl or heteroaryl, wherein the C₁₋₂₄ alkyl (e.g., C₁₋₆alkyl), or 6-24 membered aryl or heteroaryl, is optionally substitutedwith one or more aryl or heteroaryl.

For example, ring A can be

For example, ring A or B can be C₃₋₈ cycloalkyl or 3-12 memberedheterocycloalkyl.

For example, ring A or B can be piperazinyl or piperidinyl.

For example, each of R^(s1), R^(s2), and R^(s3) can be hydrogen or C₁₋₆alkyl.

In some embodiments, W^(P) is

In some embodiments, W^(P) is

In some embodiments, when W^(P) is

L^(P′) comprises

In some embodiments, when W^(P) is

In some embodiments, when W^(P) is

In some embodiments, when W^(P) is

In some embodiments, W^(P) is

In some embodiments, when W^(P) is

L^(P′) comprises

In some embodiments, W^(P) is

wherein one of X_(a) and X_(b) is H and the other is a maleimidoblocking moiety. For example, a maleimido blocking compound (i.e., acompound that can react with maleimide to convert it to succinimide) maybe used to quench the reaction between, e.g., the Linker-Drug moiety andPBRM, and a maleimido blocking moiety refers to the chemical moietyattached to the succinimide upon conversion. For example, the maleimidoblocking moieties are moieties that can be covalently attached to one ofthe two olefin carbon atoms upon reaction of the maleimido group with athiol-containing compound of Formula (II′):R₉₀—(CH₂)_(d)—SH  (II′)wherein:

R₉₀ is NHR₉₁, OH, COOR₉₃, CH(NHR₉₁)COOR₉₃ or a substituted phenyl group;

R₉₃ is hydrogen or C₁₋₄ alkyl;

R₉₁ is hydrogen, CH₃ or CH₃CO and

d is an integer from 1 to 3.

For example, the maleimido blocking compound can be cysteine, N-acetylcysteine, cysteine methyl ester, N-methyl cysteine, 2-mercaptoethanol,3-mercaptopropanoic acid, 2-mercaptoacetic acid, mercaptomethanol (i.e.,HOCH₂SH), benzyl thiol in which phenyl is substituted with one or morehydrophilic substituents, or 3-aminopropane-1-thiol. The one or morehydrophilic substituents on phenyl comprise OH, SH, methoxy, ethoxy,COOH, CHO, COC₁₋₄ alkyl, NH₂, F, cyano, SO₃H, PO₃H, and the like.

For example, the maleimido blocking group is —S—(CH₂)_(d)—R₉₀, in which,

R₉₀ is OH, COOH, or CH(NHR₉₁)COOR₉₃;

R₉₃ is hydrogen or CH₃;

R₉₁ is hydrogen or CH₃CO; and

d is 1 or 2.

For example, the maleimido blocking group is —S—CH₂—CH(NH₂)COOH.

Stretcher Unit M^(P)

M^(P), when present, is —(Z₄)-[(Z₅)-(Z₆)]_(z)—, with Z₄ connected toL^(P′) or L^(P) and Z₆ connected to L^(M); in which

z is 1, 2, or 3;

Z₄ is:

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to Z₅ or Z₆ when present or to L^(M) when Z₅ and Z₆ are bothabsent;

b₁ is an integer from 0 to 6;

e₁ is an integer from 0 to 8,

R₁₇ is C₁₋₁₀ alkylene, C₁₋₁₀ heteroalkylene, C₃₋₈ cycloalkylene, O—(C₁₋₈alkylene, arylene, —C₁₋₁₀ alkylene-arylene-, -arylene-C₁₋₁₀ alkylene-,—C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-, —(C₃₋₈ cycloalkylene-C₁₋₁₀alkylene-, 4 to 14-membered heterocycloalkylene, —C₁₋₁₀ alkylene-(4 to14-membered heterocycloalkylene)-, -(4 to 14-memberedheterocycloalkylene)-C₁₋₁₀ alkylene-, —C₁₋₁₀ alkylene-C(═O)—, —C₁₋₁₀heteroalkylene-C(═O)—, —C₃₋₈ cycloalkylene-C(═O)—, —O—(C₁₋₈alkyl)-C(═O)—, -arylene-C(═O)—, —C₁₋₁₀ alkylene-arylene-C(═O)—, -arylene—C₁₋₁₀ alkylene-C(═O)—, —C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-C(═O)—,—(C₃₋₈ cycloalkylene)-C₁₋₁₀ alkylene-C(═O)—, -4 to 14-memberedheterocycloalkylene-C(═O)—, —C₁₋₁₀ alkylene-(4 to 14-memberedheterocycloalkylene)-C(═O)—, -(4 to 14-memberedheterocycloalkylene)-C₁₋₁₀ alkylene-C(═O)—, —C₁₋₁₀ alkylene-NH—, —C₁₋₁₀heteroalkylene-NH—, —C₃₋₈ cycloalkylene-NH—, —O—(C₁₋₈ alkyl)-NH—,-arylene-NH—, —C₁₋₁₀ alkylene-arylene-NH—, -arylene-C₁₋₁₀ alkylene-NH—,—C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-NH—, —(C₃₋₈ cycloalkylene)-C₁₋₁₀alkylene-NH—, -4 to 14-membered heterocycloalkylene-NH—, —C₁₋₁₀alkylene-(4 to 14-membered heterocycloalkylene)-NH—, -(4 to 14-memberedheterocycloalkylene)-C₁₋₁₀ alkylene-NH—, —C₁₋₁₀ alkylene-S—, —C₁₋₁₀heteroalkylene-S—, —C₃₋₈ cycloalkylene-S—, —O—C₁₋₈ alkyl)-S—,-arylene-S—, —C₁₋₁₀ alkylene-arylene-S—, -arylene-C₁₋₁₀ alkylene-S—,—C₁₋₁₀ alkylene-(C₃₋₈ cycloalkylene)-S—, —(C₃₋₈ cycloalkylene)-C₁₋₁₀alkylene-S—, -4 to 14-membered heterocycloalkylene-S—, —C₁₋₁₀alkylene-(4 to 14-membered heterocycloalkylene)-S—, or -(4 to14-membered heterocycloalkylene)-C₁-C₁₀ alkylene-S—;

each Z₅ independently is absent, R₅₇—R₁₇ or a polyether unit;

each R₅₇ independently is a bond, NR₂₃, S or O;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl; and

each Z₆ independently is absent, —C₁₋₁₀ alkyl-R₃—, —C₁₋₁₀ alkyl-NR₅—,—C₁₋₁₀ alkyl-C(O)—, —C₁₋₁₀ alkyl-O—, —C₁₋₁₀ alkyl-S— or —(C₁₋₁₀alkyl-R₃)_(g1)—C₁₋₁₀ alkyl-C(O)—;

each R₃ independently is —C(O)—NR₅— or —NR₅—C(O)—;

each R₅ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, COOH, or COO—C₁₋₆ alkyl; and

g₁ is an integer from 1 to 4.

In one embodiment, Z₄ is

e.g., wherein b₁ is 1 or 4.

In another embodiment, Z₄ is

e.g., wherein b₁ is 4.

In another embodiment, Z₄ is

e.g., wherein bi is 0.

In another embodiments, Z₄ is:

In some embodiments, each Z₅ independently is a polyalkylene glycol(PAO), including but are not limited to, polymers of lower alkyleneoxides, in particular polymers of ethylene oxide, such as, for example,propylene oxide, polypropylene glycols, polyethylene glycol (PEG),polyoxyethylenated polyols, copolymers thereof and block copolymersthereof. In other embodiments, the polyalkylene glycol is a polyethyleneglycol (PEG) including, but not limited to, polydisperse PEG,monodisperse PEG and discrete PEG. Polydisperse PEGs are a heterogeneousmixture of sizes and molecular weights whereas monodisperse PEGs aretypically purified from heterogeneous mixtures and are therefore providea single chain length and molecular weight. In another embodiment, thePEG units are discrete PEGs provide a single molecule with defined andspecified chain length. In some embodiments, the polyethylene glycol ismPEG.

As used herein a subunit when referring to the PEG unit refers to apolyethylene glycol subunit having the formula

In some such embodiments, the PEG unit comprises multiple PEG subunits.

In some embodiments, when z is 2 or 3, at least one Z₅ is a polyalkyleneglycol (PAO), e.g., a PEG unit.

In one embodiment, the PEG unit comprises 1 to 6 subunits.

In another embodiment, the PEG unit comprises 1 to 4 subunits.

In other embodiments, the PEG unit comprises 1 to 3 subunits.

In one embodiment, the PEG unit comprises 2 subunits.

In another embodiment, the PEG unit comprises 1 subunit.

In other embodiments, the PEG unit comprises one or multiple PEGsubunits linked together by a PEG linking unit. The PEG linking unitthat connects one or more chains of repeating CH₂CH₂O— subunits can beZ₆. For example, Z₆ is alkyl-R₃—, —C₂₋₁₀ alkyl-NH—, —C₂₋₁₀ alkyl-C(O)—,alkyl-O— or —C₁₋₁₀ alkyl-S, wherein R₃ is —C(O)—NR₅— or —NR₅—C(O)—.

In some embodiments, the PEG linking unit is —C₁₋₁₀ alkyl-C(O)—NH— or—C₁₋₁₀ alkyl-NH—C(O)—. In one embodiment, the PEG linking unit is—(CH₂)₂—C(O)—NH—.

In some embodiments, each Z₅ is absent.

In some embodiments, when z is 2 or 3, at least one Z₅ is absent.

In some embodiments, each Z₅ is —(CH₂—CH₂—O—)₂—.

In some embodiments, when z is 2 or 3, at least one Z₅ is—(CH₂—CH₂—O—)₂—.

In some embodiments, each Z₅ independently is R₅₇—R₁₇. For example, eachZ₅ independently is R₁₇, NHR₁₇, OR₁₇, or SR₁₇.

In some embodiments, when z is 2 or 3, at least one Z₅ is R₅₇—R₁₇, e.g.,R₁₇, NHR₁₇, OR₁₇, or SR₁₇.

In some embodiments, each Z₆ is absent.

In some embodiments, when z is 2 or 3, at least one Z₆ is absent.

In some embodiments, at least one of Z₅ and Z₆ is not absent.

In some embodiments, each Z₆ independently is —C₁₋₁₀ alkyl-R₃—, —C₁₋₁₀alkyl-NH—, alkyl-C(O)—, —C₁₋₁₀ alkyl-O—, —C₁₋₁₀ alkyl-S— or —(C₁₋₁₀alkyl-R₃)_(g1)—C₁₋₁₀ alkyl-C(O)—. For example, g₁ is an integer from 1to 4.

In some embodiments, when z is 2 or 3, at least one Z₆ is —C₁₋₁₀alkyl-R₃—, —C₁₋₁₀ alkyl-NH—, —C₁₋₁₀ alkyl-C(O)—, —C₁₋₁₀ alkyl-O—,alkyl-S— or —(C₁₋₁₀ alkyl-R₃)_(g1)—C₁₋₁₀ alkyl-C(O)—. For example, g₁ isan integer from 1 to 4.

In some embodiments, each Z₆ independently or at least one Z₆ is —C₂₋₁₀alkyl-C(O)—, e.g., —(CH₂)₂—C(O)—.

In some embodiments, each Z₆ independently or at least one Z₆ is —C₂₋₁₀alkyl-R₃—C₂₋₁₀ alkyl-C(O)—, e.g., —(CH₂)₂—C(O)NH—(CH₂)₂—C(O)—.

In some embodiments, each Z₆ independently or at least one Z₆ is —(C₂₋₁₀alkyl-R₃)_(g1)—C₂₋₁₀ alkyl-C(O)—, e.g.,—(CH₂)₂—C(O)NH—(CH₂)₂—NHC(O)—(CH₂)—C(O)—.

In one embodiment, —[(Z₅)-(Z₆)]_(z)— is not absent.

In one embodiment, —[(Z₅)-(Z₆)]_(z)— is a bond.

In one embodiment, —[(Z₅)-(Z₆)]_(z) is—(CH₂CH₂O)₂—(CH₂)₂—C(O)—NH—(CH₂CH₂O)₂—.

In some embodiments, M^(P), when present, is

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to L^(M);

R₃, R₅, R₁₇, and R₂₃ are as defined herein;

R₄ is a bond or —NR₅—(CR₂₀R₂₁)—C(O)—;

each R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

each b₁ independently is an integer from 0 to 6;

e₁ is an integer from 0 to 8,

each f₁ independently is an integer from 1 to 6; and

g₂ is an integer from 1 to 4.

In some embodiments, b₁ is 1.

In some embodiments, each f₁ independently is 1 or 2.

In some embodiments, f₁ is 2.

In some embodiments, g₂ is 1 or 2.

In some embodiments, g₂ is 2.

In some embodiments, R₁₇ is unsubstituted.

In some embodiments, R₁₇ is optionally substituted.

In some embodiments, R₁₇ is optionally substituted by a basic unit,e.g., —(CH₂)_(x)NH₂, —(CH₂)_(x)NHR^(a), and —(CH₂)_(x)N(R^(a))₂, whereinx is an integer from 1 to 4 and each R^(a) is independently selectedfrom C₁₋₆ alkyl and C₁₋₆ haloalkyl, or two R^(a) groups are combinedwith the nitrogen to which they are attached to form an azetidinyl,pyrrolidinyl or piperidinyl group.

In some embodiments, R¹⁷ is —C₂₋₅ alkylene-C(═O)— wherein the alkyleneis optionally substituted by a basic unit, e.g., —(CH₂)_(x)NH₂,—(CH₂)_(x)NHR^(a), and —(CH₂)_(x)N(R^(a))₂, wherein x and R^(a) are asdefined herein.

In some embodiments, wherein M^(P), when present is:

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to L^(M).

In some embodiments, wherein M^(P), when present, is:

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to L^(M).

In some embodiments, wherein M^(P), when present, is:

wherein * denotes attachment to L^(P′) or L^(P) and ** denotesattachment to L^(M).L^(M) and W^(M)

L^(M) is a bond, or a multi-armed linker (e.g., trivalent or tetravalentor having 3 or 4 arms), wherein each arm maybe the same or different.

In some embodiments, a₂ is 2 and L^(M) is

wherein:

denotes attachment to M^(P) when present or attachment to L^(P) orL^(P′) when M^(P) is absent;

Y₁ denotes attachment to L³ when present or attachment to M^(A) when L³is absent;

R₂ and R′₂ are each independently hydrogen, an optionally substitutedC₁₋₆ alkyl, an optionally substituted C₂₋₆ alkenyl, an optionallysubstituted C₂₋₆ alkynyl, an optionally substituted C₃₋₁₉ branchedalkyl, an optionally substituted C₃₋₈ cycloalkyl, an optionallysubstituted C₆₋₁₀ aryl, an optionally substituted heteroaryl, anoptionally substituted C₁₋₆ heteroalkyl, C₁₋₆ alkoxy, aryloxy, C₁₋₆heteroalkoxy, C₂₋₆ alkanoyl, an optionally substituted arylcarbonyl,C₂₋₆ alkoxycarbonyl, C₂₋₆ alkanoyloxy, arylcarbonyloxy, an optionallysubstituted C₂₋₆ alkanoyl, an optionally substituted C₂₋₆ alkanoyloxy,an optionally substituted C₂₋₆ substituted alkanoyloxy, —COOH, or—COO—C₁₋₆ alkyl;

each of c₁, c₂, c₃, c₄, c₅, c₇, and c₈ is an integer independentlyranging between 0 and 10; and

each of d₁, d₂, d₃, d₄, d₅, and d₇ is an integer independently rangingbetween 0 and 10.

In some embodiments, a₂ is 2 and L^(M) is

In some embodiments, c₁, c₂, c₃, c₄, c₅, c₇, and c₈ are eachindependently 0 or 1.

In some embodiments, c₁, c₂, c₃, c₄, c₅, c₇, and c₈ are eachindependently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, c₁, c₂, c₃, c₄, c₅, c₇, and c₈ are eachindependently 0, 1 or 2.

In some embodiments, d₁, d₂, d₃, d₄, d₅, and d₇ are each independently 0or 1.

In some embodiments, d₁, d₂, d₃, d₄, d₅, and d₇ are each independently1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, d₁, d₂, d₃, d₄, d₅, and d₇ are each independently1, 2, 3 or 4.

In some embodiments, R₂ and R′₂ are each independently hydrogen, C₁₋₆alkyl, C₆₋₁₀ aryl, C₃₋₈ cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

In some embodiments, R₂ and R′₂ are each independently hydrogen or C₁₋₆alkyl.

In some embodiments, R₂ and R′₂ are each independently hydrogen.

In some embodiments, R₂ and R′₂ are each independently C₁₋₆ alkyl.

In some embodiments, L^(M) is:

In some embodiments, a₂ is 3 and L^(M) is

wherein:

denotes attachment to M^(P) when present or attachment to L^(P) orL^(P′) when M^(P) is absent;

Y₁ denotes attachment to L³ when present or attachment to M^(A) when L³is absent;

R₂ and R′₂ are each independently hydrogen, an optionally substitutedC₁₋₆ alkyl, an optionally substituted C₂₋₆ alkenyl, an optionallysubstituted C₂₋₆ alkynyl, an optionally substituted C₃₋₁₉ branchedalkyl, an optionally substituted C₃₋₈ cycloalkyl, an optionallysubstituted C₆₋₁₀ aryl, an optionally substituted heteroaryl, anoptionally substituted C₁₋₆ heteroalkyl, C₁₋₆ alkoxy, aryloxy, C₁₋₆heteroalkoxy, C₂₋₆ alkanoyl, an optionally substituted arylcarbonyl,C₂₋₆ alkoxycarbonyl, C₂₋₆ alkanoyloxy, arylcarbonyloxy, an optionallysubstituted C₂₋₆ alkanoyl, an optionally substituted C₂₋₆ alkanoyloxy,an optionally substituted C₂₋₆ substituted alkanoyloxy, —COOH, or—COO—C₁₋₆ alkyl;

each of c₁, c₂, c₃, c₄, c₅, c₆, c₇, and c₈ is an integer independentlyranging between 0 and 10;

each of d₁, d₂, d₃, d₄, d₅, d₆, d₇ and d₈ is an integer independentlyranging between 0 and 10; and

each of e₁, e₂, e₃, e₄, e₅, e₆, e₇, and e₈ is an integer independentlyranging between 0 and 10.

In some embodiments, a₂ is 3 and L^(M) is

In some embodiments, -L^(M)-(L³)_(a2)- is:

In certain embodiments, a₂ is 2 and L^(M) is selected from

wherein:

the wavy line indicates attachment sites within the conjugate of thedisclosure or intermediates thereof;

R₁₁₀ is:

wherein the asterisk indicates attachment to the carbon labeled x andthe wavy line indicates one of the three attachment sites;

R₁₀₀ is independently selected from hydrogen or —C₁₋₃ alkyl;

Y is N or CH;

each occurrence of Y′ is independently selected from NH, O, or S, and

each occurrence of c′ is independently an integer from 1 to 10.

In some embodiments, R₁₀₀ is independently selected from hydrogen andCH₃.

In some embodiments, Y is N.

In some embodiments, Y is CH.

In some embodiments, R₁₀₀ is H or CH₃.

In some embodiments, each c′ is independently an integer from 1 to 3.

In some embodiments, R₁₁₀ is not

In some embodiments, where an AA unit has two attachment sites (i.e., aterminal drug unit) one of the attachment sites shown above canreplaced, for example, by H, OH, or a C₁₋₃ unsubstituted alkyl group.

When L^(M) is a multi-armed linker and not yet connected to theStretcher unit M^(P), W^(M) is a terminus of L^(M) and each occurrenceof W^(M) is independently hydrogen, a protecting group, a leaving group,or a functional group that is capable of connecting L^(M) to M^(P) byforming a covalent bond.

In some embodiments, W^(M) is an amine protecting group, e.g., BOC. Insome embodiments, W^(M) is an amine protecting group, e.g., BOC, andL^(M) is

In some embodiments, W^(M) comprises an amine group, e.g.,—C(O)—(CH₂)_(w)—NH₂, in which w is an integer from 1 to 6.

In some embodiments, W^(M) is —C(O)—CH₂—NH₂. In some embodiments, W^(M)is —C(O)—CH₂—NH₂ and L^(M) is

In some embodiments, W^(M) is hydrogen.

M^(A)

M^(A) is a linker moiety that is capable of connecting one or more drugsand one or more hydrophilic groups to L^(P) or L^(P′). In someembodiments, M^(A) comprises a peptide moiety of at least two aminoacids (AA's).

The peptide moiety is a moiety that is capable of forming a covalentbond with a -L^(D)-D unit and allows for the attachment of multipledrugs. In some embodiments, peptide moiety comprises a single AA unit orhas two or more AA units (e.g., 2 to 10, preferably from 2 to 6, e.g.,2, 3, 4, 5 or 6) wherein the AA units are each independently a naturalor non-natural amino acid, an amino alcohol, an amino aldehyde, adiamine, or a polyamine or combinations thereof. If necessary in orderto have the requisite number of attachments, at least one of AA unitswill have a functionalized side chain to provide for attachment of the-L^(D)-D unit. Exemplary functionalized AA units (e.g., amino acids,amino alcohols, or amino aldehydes) include, for example, azido oralkyne functionalized AA units (e.g., amino acid, amino alcohol, oramino aldehyde modified to have an azide group or alkyne group forattachment using click chemistry).

In some embodiments, the peptide moiety has 2 to 12 AA units.

In some embodiments, the peptide moiety has 2 to 10 AA units.

In another embodiment, the peptide moiety has 2 to 6 AA units.

In yet another embodiment, the peptide moiety has 2, 3, 4, 5 or 6 AAunits.

In some embodiments, an AA unit has three attachment sites, (e.g., forattachment to L^(M), the hydrophilic group or another AA unit, and tothe -L^(D)-D unit). For example, the AA unit has the formula below:

wherein the wavy line indicates attachment sites within the conjugate ofthe disclosure or intermediates thereof; and R₁₀₀ and R₁₁₀ are asdefined herein.

In some embodiments, an AA unit has two attachment sites (i.e., aterminal unit) and one of the attachment sites shown above can replaced,for example, by H, OH, or an unsubstituted C₁₋₃ alkyl group.

In some embodiments, the peptide moiety comprises at least two AA unitsof the following formula:

wherein:

each R₁₁₁ independently is H, p-hydroxybenzyl, methyl, isopropyl,isobutyl, sec-butyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂,—CH₂COOH, —CH₂CH2CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl,

the wavy line indicates the attachment sites within the conjugate orintermediates thereof; and

R₁₀₀ and R₁₁₀ are as defined herein.

In some embodiments, the peptide moiety comprises at least two AA units,e.g., cysteine-alanine as shown below:

wherein the wavy lines and asterisk indicates attachment sites withinthe conjugate or intermediates thereof. For example, asterisk indicatesattachment site of -L^(D)-D unit or a hydrophilic group. For example,the wavy line next to the carbonyl group indicates attachment site of-L^(D)-D unit or a hydrophilic group. For example, the wavy line next tothe amine group indicates attachment site of -L^(D)-D unit or ahydrophilic group. For example, one or two of the wavy lines andasterisk indicate attachment site(s) of one or more -L^(D)-D units orone or more hydrophilic groups.

In some embodiments, the peptide moiety comprises at least two AA units,which provide two attachment sites, e.g., cysteine-alanine as shownbelow:

wherein the wavy line and asterisk indicates attachment sites within theconjugate or intermediates thereof. For example, asterisk indicatesattachment site of -L^(D)-D unit or a hydrophilic group. For example,the wavy line indicates attachment site of -L^(D)-D unit or ahydrophilic group.

One or more AA units (e.g., an amino acid, amino alcohol, amino aldehydeor polyamine) of the peptide moiety can be replaced by an optionallysubstituted C₁₋₂₀ heteroalkylene (e.g., optionally substituted C₁₋₁₂heteroalkylene), optionally substituted C₃₋₈ heterocyclo, optionallysubstituted C₆₋₁₄ arylene, or optionally substituted C₃₋₈ carbocyclo asdescribed herein. The optionally substituted heteroalkylene,heterocycle, arylene or carbocyclo may have one or more functionalgroups for attachment within a conjugate or intermediates thereof.Suitable substituents include, but are not limited to (═O), —R^(1C),—R^(1B), —OR^(1B), —SR^(1B), —N(R^(1B))₂, —N(R^(1B))₃, ═NR^(1B),C(R^(1C))₃, CN, OCN, SCN, N═C═O, NCS, NO, NO₂, ═N₂, N₃,NR^(1B)C(═O)R^(1B), —C(═O)R^(1B), —C(═O)N(R^(1B))₂, SO₃ ⁻, SO₃H,S(═O)₂R^(1B), —OS(═O)₂OR^(1B), —S(═O)₂NR^(1B), —S(═O)R^(1B),—OP(═O)(OR^(1B))₂, —P(═O)(OR^(1B))₂, PO₃ ⁻; PO₃H₂, AsO₂H₂, C(═O)R^(1B),C(═O)R^(1C), C(═S)R^(1B), CO₂R^(1B), CO₂—, C(═S)OR^(1B), C(═O)SR^(1B),C(═S)SR^(1B), C(═O)N(R^(1B))₂, C(═S)N(R^(1B))₂, andC(═NR^(1B))N(R^(1B))₂, where each R^(1C) is independently a halogen(e.g., —F, —Cl, —Br, or —I), and each R^(1B) is independently —H, —C₁₋₂₀alkyl, —C₆₋₂₀ aryl, —C₃₋₁₄ heterocycle, a protecting group or a prodrugmoiety.

In some embodiments, the one or more substituents for theheteroalkylene, heterocycle, arylene or carbocyclo are selected from(═O), R^(1C), R^(1B), OR^(1B), SR^(1B), and N(R^(1B))₂.

In some embodiments, the peptide moiety can be a straight chain orbranched moiety of having the Formula:

wherein:

each BB′ is independently an amino acid, optionally substituted C₁₋₂₀heteroalkylene (e.g., optionally substituted C₁₋₁₂ heteroalkylene),optionally substituted C₃₋₈ heterocyclo, optionally substituted C₆₋₁₄arylene, or optionally substituted C₃-C₈ carbocyclo;

d₁₂ is an integer from 1 to 10; and

the wavy line indicates the covalent attachment sites within theconjugate or intermediate thereof.

In some embodiments, d₁₂ is an integer from 2 to 10.

In some embodiments, d₁₂ is an integer from 2 to 6.

In some embodiments, d₁₂ is an integer from 4, 5 or 6.

In some embodiments, d₁₂ is an integer from 5 or 6.

In some embodiments, the optionally substituted heteroalkylene,heterocycle, arylene or carbocyclo have functional groups forattachments between the BB′ subunits and/or for attachments within aconjugate or intermediates thereof disclosed herein.

In some embodiments, the peptide moiety comprises no more than 2optionally substituted C₁₋₂₀ heteroalkylenes, optionally substitutedC₃₋₁₈ heterocyclos, optionally substituted C₆₋₁₄ arylenes, or optionallysubstituted C₃₋₈ carbocyclos.

In other embodiments, the peptide moiety comprises no more than 1optionally substituted C₁₋₂₀ heteroalkylenes, optionally substitutedC₃₋₁₈ heterocyclos, optionally substituted C₆₋₁₄ arylenes, or optionallysubstituted C₃₋₈ carbocyclos. The optionally substituted heteroalkylene,heterocycle, arylene or carbocyclo will have functional groups forattachment between the BB′ subunits and/or for attachments within aconjugate or intermediates thereof disclosed herein.

In some embodiments, at least one BB′ is an amino acid. For example, theamino acid can be an alpha, beta, or gamma amino acid, which can benatural or non-natural. The amino acid can be a D or L isomer.

In some embodiments, attachment within the peptide moiety or with theother components of the conjugate (or intermediate thereof, or scaffold)can be, for example, via amino, carboxy, or other functionalities.

In one embodiment, each amino acid of the peptide moiety can beindependently D or L isomer of a thiol containing amino acid. The thiolcontaining amino acid can be, for example, cysteine, homocysteine, orpenicillamine.

In another embodiment, each amino acid that comprises the peptide moietycan be independently the L- or D-isomers of the following amino acids:alanine (including β-alanine), arginine, aspartic acid, asparagine,cysteine, histidine, glycine, glutamic acid, glutamine, phenylalanine,lysine, leucine, methionine, serine, tyrosine, threonine, tryptophan,proline, ornithine, penicillamine, aminoalkynoic acid, aminoalkanedioicacid, heterocyclo-carboxylic acid, citrulline, statine, diaminoalkanoicacid, stereoisomers thereof (e.g., isoaspartic acid and isoglutamicacid), and derivatives thereof.

In one embodiment, each amino acid that comprises the peptide moiety isindependently cysteine, homocysteine, penicillamine, ornithine, lysine,serine, threonine, glycine, glutamine, alanine, aspartic acid, glutamicacid, selenocysteine, proline, glycine, isoleucine, leucine, methionine,valine, alanine, or a stereoisomers thereof (e.g., isoaspartic acid andisoglutamic acid).

In some embodiments, the peptide moiety comprises a monopeptide, adipeptide, tripeptide, tetrapeptide, or pentapeptide.

In some embodiments, the peptide moiety contains at least about fiveamino acids (e.g., 5, 6, 7, 8, 9, or 10 amino acids).

In some embodiments, the peptide moiety contains at most about ten aminoacids.

In one embodiment, the peptide moiety comprises a pentapeptide.

In one embodiment, each amino acid that comprises the peptide moiety isindependently glycine, serine, glutamic acid, lysine, aspartic acid andcysteine.

In another embodiment, the peptide moiety comprises at least fourglycines and at least one serine, e.g., (glycine)₄ and serine whereinthe serine is at any position along the peptide chain, such as, forexample, (serine)-(glycine)₄; (glycine)-(serine)-(glycine)₃;(glycine)₂-(serine)-(glycine)₂; (glycine)₃-(serine)-(glycine); or(glycine)₄-(serine).

In another embodiment, the peptide moiety comprises (glycine)₄-(serine)or (serine)-(glycine)₄.

In another embodiment, the peptide moiety comprises at least fourglycines and at least one glutamic acid e.g., (glycine)₄ and glutamicacid wherein the glutamic acid is at any position along the peptidechain, such as, for example, (glutamic acid)-(glycine)₄;(glycine)-(glutamic acid)-(glycine)₃; (glycine)₂-(glutamicacid)-(glycine)₂; (glycine)₃-(glutamic acid)-(glycine); or(glycine)₄-(glutamic acid).

In another embodiment, the peptide moiety comprises (glutamicacid)-(glycine)₄; or (glycine)₄-(glutamic acid).

In another embodiment, the peptide moiety comprises(β-alanine)-(glycine)₄-(serine) wherein the serine is at any positionalong the peptide chain, such as, for example,(β-alanine)-(serine)-(glycine)₄;(β-alanine)-(glycine)-(serine)-(glycine)₃;(β-alanine)-(glycine)₂-(serine)-(glycine)₂;(β-alanine)-(glycine)₃-(serine)-(glycine); or(β-alanine)-(glycine)₄-(serine).

In another embodiment, the peptide moiety comprises(glycine)₄-(serine)-(glutamic acid) wherein the serine is at anyposition along the peptide chain, such as, for example,(serine)-(glycine)₄-(glutamic acid);(glycine)-(serine)-(glycine)₃-(glutamic acid);(glycine)₂-(serine)-(glycine)₂-(glutamic acid);(glycine)₃-(serine)-(glycine)-(glutamic acid); or(glycine)₄-(serine)-(glutamic acid). In another embodiment, the peptidemoiety comprises (β-alanine)-(glycine)₄-(serine)-(glutamic acid) whereinthe serine is at any position along the peptide chain, such as, forexample, (β-alanine)-(serine)-(glycine)₄-(glutamic acid);(β-alanine)-(glycine)-(serine)-(glycine)₃-(glutamic acid);(β-alanine)-(glycine)₂-(serine)-(glycine)₂-(glutamic acid);(β-alanine)-(glycine)₃-(serine)-(glycine)-(glutamic acid); or(β-alanine)-(glycine)₄-(serine)-(glutamic acid).

In other embodiments, when at least one of hydrophilic groups (or T¹) isa polyalcohol or derivative thereof (e.g., an amino polyalcohol) or aglucosyl-amine or a di-glucosyl-amine or a tri-glucosyl-amine, M^(A)does not have to comprise a peptide moiety, e.g., M^(A) comprising thosemulti-armed linkers as listed herein for L^(M). For example, M^(A)comprises one or more of the following:

wherein:

the wavy line indicates attachment sites within the conjugate of thedisclosure or intermediates thereof; and R₁₀₀ and R₁₁₀ are as definedherein.

In some embodiments, R₁₁₀ is:

wherein the asterisk indicates attachment to the carbon labeled x andthe wavy line indicates one of the three attachment sites.

In some embodiments, R₁₀₀ is independently selected from hydrogen andCH₃.

In some embodiments, Y is N.

In some embodiments, Y is CH.

In some embodiments, R₁₀₀ is H or CH₃.

In some embodiments, each c′ is independently an integer from 1 to 3.

In some embodiments, R₁₁₀ is not

L^(D) and W^(D)

Each occurrence of L^(D) is independently a divalent linker moietyconnecting D to M^(A) and comprises at least one cleavable bond suchthat when the bond is broken, D is released in an active form for itsintended therapeutic effect.

In some embodiments, L^(D) is a component of the Releasable AssemblyUnit. In other embodiments, L^(D) is the Releasable Assembly Unit.

In some embodiments, L^(D) comprises one cleavable bond.

In some embodiments, L^(D) comprises multiple cleavage sites or bonds.

Functional groups for forming a cleavable bond can include, for example,sulfhydryl groups to form disulfide bonds, aldehyde, ketone, orhydrazine groups to form hydrazone bonds, hydroxylamine groups to formoxime bonds, carboxylic or amino groups to form peptide bonds,carboxylic or hydroxy groups to form ester bonds, and sugars to formglycosidic bonds. In some embodiments, comprises a disulfide bond thatis cleavable through disulfide exchange, an acid-labile bond that iscleavable at acidic pH, and/or bonds that are cleavable by hydrolases(e.g., peptidases, esterases, and glucuronidases). In some embodiments,comprises a carbamate bond (i.e., —O—C(O)—NR—, in which R is H or alkylor the like).

The structure and sequence of the cleavable bond(s) in L^(D) can be suchthat the bond(s) is cleaved by the action of enzymes present at thetarget site. In other embodiments, the cleavable bond(s) can becleavable by other mechanisms.

In some embodiments, the cleavable bond(s) can be enzymatically cleavedby one or more enzymes, including a tumor-associated protease, toliberate the Drug unit or D, which in one embodiment is protonated invivo upon release to provide a Drug unit or D.

In certain embodiments, L^(D) can comprise one or more amino acids. Forexample, each amino acid in L^(D) can be natural or unnatural and/or aD- or L-isomer provided that there is a cleavable bond. In someembodiments, L^(D) comprising an alpha, beta, or gamma amino acid thatcan be natural or non-natural. In some embodiments, L^(D) comprises 1 to12 (e.g., 1 to 6, or 1 to 4, or 1 to 3, or 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, or 12) amino acids in contiguous sequence.

In certain embodiments, L^(D) can comprise only natural amino acids. Inother embodiments, L^(D) can comprise only non-natural amino acids. Insome embodiments, L^(D) can comprise a natural amino acid linked to anon-natural amino acid. In some embodiments, L^(D) can comprise anatural amino acid linked to a D-isomer of a natural amino acid. Anexemplary L^(D) comprises a dipeptide such as -Val-Cit-, -Phe-Lys- or-Val-Ala-.

In some embodiments, L^(D) comprises, a monopeptide, a dipeptide, atripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, aheptapeptide, an octapeptide, a nonapeptide, a decapeptide, anundecapeptide or a dodecapeptide unit.

In some embodiments, L^(D) comprises a peptide (e.g., of 1 to 12 aminoacids), which is conjugated directly to the drug unit. In some suchembodiments, the peptide is a single amino acid or a dipeptide.

In some embodiments, each amino acid in L^(D) is independently selectedfrom alanine, β-alanine, arginine, aspartic acid, asparagine, histidine,glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine,serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine,cysteine, methionine, selenocysteine, ornithine, penicillamine,aminoalkanoic acid, aminoalkynoic acid, aminoalkanedioic acid,aminobenzoic acid, amino-heterocyclo-alkanoic acid,heterocyclo-carboxylic acid, citrulline, statine, diaminoalkanoic acid,and derivatives thereof.

In some embodiments, each amino acid is independently selected fromalanine, β-alanine, arginine, aspartic acid, asparagine, histidine,glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine,serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine,cysteine, methionine, citrulline and selenocysteine.

In some embodiments, each amino acid is independently selected from thegroup consisting of alanine, β-alanine, arginine, aspartic acid,asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine,lysine, leucine, serine, tyrosine, threonine, isoleucine, proline,tryptophan, valine, citrulline and derivatives thereof.

In some embodiments, each amino acid is selected from the proteinogenicor the non-proteinogenic amino acids.

In some embodiments, each amino acid in L^(D) can be independentlyselected from L- or D-isomers of the following amino acids: alanine,β-alanine, arginine, aspartic acid, asparagine, cysteine, histidine,glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine,methionine, serine, tyrosine, threonine, tryptophan, proline, ornithine,penicillamine, aminoalkynoic acid, aminoalkanedioic acid,heterocyclo-carboxylic acid, citrulline, statine, diaminoalkanoic acid,valine, citrulline or derivatives thereof.

In some embodiments, each amino acid in L^(D) is independently cysteine,homocysteine, penicillamine, ornithine, lysine, serine, threonine,glycine, glutamine, alanine, aspartic acid, glutamic acid,selenocysteine, proline, glycine, isoleucine, leucine, methionine,valine, citrulline or alanine.

In some embodiments, each amino acid in L^(D) is independently selectedfrom L-isomers of the following amino acids: alanine, β-alanine,arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid,glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine,isoleucine, tryptophan, citrulline or valine.

In some embodiments, each amino acid in L^(D) is independently selectedfrom D-isomers of the following amino acids: alanine, β-alanine,arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid,glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine,isoleucine, tryptophan, citrulline or valine.

In some embodiments, each amino acid in L^(D) is alanine, β-alanine,glutamic acid, isoglutamic acid, isoaspartic acid, valine citrulline oraspartic acid.

In one embodiment, comprises β-alanine.

In another embodiment, L^(D) comprises (β-alanine)-(alanine).

In another embodiment, comprises (β-alanine)-(glutamic acid).

In another embodiment, L^(D) comprises (β-alanine)-(isoglutamic acid).

In another embodiment, L^(D) comprises (β-alanine)-(aspartic acid).

In another embodiment, L^(D) comprises (β-alanine)-(isoaspartic acid).

In another embodiment, L^(D) comprises (β-alanine)-(valine).

In another embodiment, L^(D) comprises (β-alanine)-(valine)-(alanine).

In another embodiment, L^(D) comprises (β-alanine)-(alanine)-(alanine).

In another embodiment, L^(D) comprises (β-alanine)-(valine)-(citruline).

In some embodiments, L^(D) comprises a carbamate bond in addition to oneor more amino acids.

In certain embodiments, L^(D) can be designed and optimized in theirselectivity for enzymatic cleavage by a particular enzyme, e.g., atumor-associated protease.

In one embodiment, L^(D) comprises a bond whose cleavage is catalyzed bycathepsin B, C and D, or a plasmin protease.

In another embodiment, L^(D) comprises a sugar cleavage site. In somesuch embodiments, L^(D) comprises a sugar moiety (Su) linked via anoxygen glycosidic bond to a self-immolative group. A “self-immolativegroup” can be a tri-functional chemical moiety that is capable ofcovalently linking together three spaced chemical moieties (i.e., thesugar moiety (via a glycosidic bond), a drug unit (directly orindirectly), and M^(A) (directly or indirectly). The glycosidic bondwill be one that can be cleaved at the target site to initiate aself-immolative reaction sequence that leads to a release of the drug.

For example, L^(D) comprises a sugar moiety (Su) linked via a glycosidebond (—O′—) to a self-immolative group (K) of the formula:

wherein the self-immolative group (K) forms a covalent bond with thedrug unit (directly or indirectly) and also forms a covalent bond withM^(A) (directly or indirectly). Examples of self-immolative groups aredescribed in, e.g., WO 2015/057699, the contents of which are herebyincorporated by reference in its entirety.

When not connected to or prior to connecting to a drug, L^(D) comprisesa functional a functional group W^(D). Each W^(D) independently can be afunctional group as listed for W^(P). For example, each W^(D)independently is

in which R^(1A) is a sulfur protecting group, each of ring A and B,independently, is cycloalkyl or heterocycloalkyl, R^(W) is an aliphatic,heteroaliphatic, carbocyclic or heterocycloalkyl moiety; ring D isheterocycloalkyl; R^(1J) is hydrogen, an aliphatic, heteroaliphatic,carbocyclic, or heterocycloalkyl moiety; and R^(1K) is a leaving group(e.g., halide or RC(O)O— in which R is hydrogen, an aliphatic,heteroaliphatic, carbocyclic, or heterocycloalkyl moiety).

In some embodiments, W^(D) is

In some embodiments, W^(D) is

wherein one of X_(a) and X_(b) is H and the other is a maleimidoblocking moiety.

In some embodiments, W^(D) is

Therapeutic Agents, Drug Unit, or D

In certain embodiments, the therapeutic agent is a small molecule havinga molecular weight preferably ≤about 5 kDa, more preferably ≤about 4kDa, more preferably ≤about 3 kDa, most preferably ≤about 1.5 kDa or≤about 1 kDa.

In certain embodiments, the therapeutic agent has an IC₅₀ of about lessthan 1 nM.

In another embodiment, the therapeutic agent has an IC₅₀ of aboutgreater than 1 nM, for example, the therapeutic agent has an IC₅₀ ofabout 1 to 50 nM.

Some therapeutic agents having an IC₅₀ of greater than about 1 nM (e.g.,“less potent drugs”) are unsuitable for conjugation with an antibodyusing art-recognized conjugation techniques. Without wishing to be boundby theory, such therapeutic agents have a potency that is insufficientfor use in targeted antibody-drug conjugates using conventionaltechniques as sufficient copies of the drug (i.e., more than 8) cannotbe conjugated using art-recognized techniques without resulting indiminished pharmacokinetic and physiochemical properties of theconjugate. However sufficiently high loadings of these less potent drugscan be achieved using the conjugation strategies described hereinthereby resulting in high loadings of the therapeutic agent whilemaintaining the desirable pharmacokinetic and physiochemical properties.Thus, the disclosure also relates to an antibody-drug conjugate whichincludes an antibody, a scaffold and at least eight therapeutic agentmoieties, wherein the therapeutic agent has an IC₅₀ of greater thanabout 1 nM.

The small molecule therapeutic agents used in this disclosure (e.g.,antiproliferative (cytotoxic and cytostatic) agents capable of beinglinked to a targeting moiety via the linker(s) of the disclosure)include cytotoxic compounds (e.g., broad spectrum), angiogenesisinhibitors, cell cycle progression inhibitors, PI3K/m-TOR/AKT pathwayinhibitors, MAPK signaling pathway inhibitors, kinase inhibitors,protein chaperones inhibitors, HDAC inhibitors, PARP inhibitors,nicotinamide phosphoribosyl transferase (NAMPT) inhibitors, Wnt/Hedgehogsignaling pathway inhibitors and RNA polymerase inhibitors.

Broad spectrum cytotoxins include, but are not limited to, DNA-binding,intercalating or alkylating drugs, microtubule stabilizing anddestabilizing agents, platinum compounds, topoisomerase I inhibitors andprotein synthesis inhibitors.

Exemplary DNA-binding, intercalation or alkylating drugs include,CC-1065 and its analogs, anthracyclines (doxorubicin, epirubicin,idarubicin, daunorubicin, nemorubicin and its derivatives, PNU-159682),bisnapththalimide compounds such as elinafide (LU79553) and its analogs,alkylating agents, such as calicheamicins, dactinomycins, mitomycins,pyrrolobenzodiazepines, and the like. Exemplary CC-1065 analogs includeduocarmycin SA, duocarmycin A, duocarmycin C1, duocarmycin C2,duocarmycin B1, duocarmycin B2, duocarmycin D, DU-86, KW-2189,adozelesin, bizelesin, carzelesin, seco-adozelesin, and related analogsand prodrug forms, examples of which are described in U.S. Pat. Nos.5,475,092; 5,595,499; 5,846,545; 6,534,660; 6,586,618; 6,756,397 and7,049,316. Doxorubicin and its analogs include those described in U.S.Pat. No. 6,630,579. Calicheamicins include, e.g., enediynes, e.g.,esperamicin, and those described in U.S. Pat. Nos. 5,714,586 and5,739,116. Duocarmycins include those described in U.S. Pat. Nos.5,070,092; 5,101,038; 5,187,186; 6,548,530; 6,660,742; and 7,553,816 B2;and Li et al., Tet Letts., 50:2932-2935 (2009).

Pyrrolobenzodiazepines (PBD) and analogs thereof include those describedin Denny, Exp. Opin. Ther. Patents., 10(4):459-474 (2000) and Antonowand Thurston, Chem Rev., 2815-2864 (2010).

Exemplary microtubule stabilizing and destabilizing agents includetaxane compounds, such as paclitaxel, docetaxel, tesetaxel andcarbazitaxel; maytansinoids, auristatins and analogs thereof, vincaalkaloid derivatives, epothilones and cryptophycins.

Exemplary maytansinoids or maytansinoid analogs include maytansinol andmaytansinol analogs, maytansine or DM-1 and DM-4 are those described inU.S. Pat. Nos. 5,208,020; 5,416,064; 6,333.410; 6,441,163; 6,716,821;RE39,151 and 7,276,497. In certain embodiments, the cytotoxic agent is amaytansinoid, another group of anti-tubulin agents (ImmunoGen, Inc.; seealso Chari et al., 1992, Cancer Res. 52:127-131), maytansinoids ormaytansinoid analogs. Examples of suitable maytansinoids includemaytansinol and maytansinol analogs. Suitable maytansinoids aredisclosed in U.S. Pat. Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016;4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866;4,450,254; 4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and5,846,545.

Exemplary auristatins include auristatin E (also known as a derivativeof dolastatin-10), auristatin EB (AEB), auristatin EFP (AEFP),monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF),auristatin F, auristatin F phenylenediamine (AFP), auristatin Fhydroxylpropylamide (AF HPA), monomethyl auristatin Fhydroxylpropylamide (MMAF HPA), and dolastatin. Suitable auristatins arealso described in U.S. Publication Nos. 2003/0083263, 2011/0020343, and2011/0070248; PCT Application Publication Nos. WO 09/117531, WO2005/081711, WO 04/010957; WO 02/088172 and WO 01/24763, and U.S. Pat.Nos. 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,124,431; 6,034,065;5,780,588; 5,767,237; 5,665,860; 5,663,149; 5,635,483; 5,599,902;5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036;5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414,the disclosures of which are incorporated herein by reference in theirentirety.

Exemplary vinca alkaloids include vincristine, vinblastine, vindesine,and navelbine (vinorelbine). Suitable Vinca alkaloids that can be usedin the present disclosure are also disclosed in U.S. Publication Nos.2002/0103136 and 2010/0305149, and in U.S. Pat. No. 7,303,749 B1, thedisclosures of which are incorporated herein by reference in theirentirety.

Exemplary epothilone compounds include epothilone A, B, C, D, E and F,and derivatives thereof. Suitable epothilone compounds and derivativesthereof are described, for example, in U.S. Pat. Nos. 6,956,036;6,989,450; 6,121,029; 6,117,659; 6,096,757; 6,043,372; 5,969,145; and5,886,026; and WO 97/19086; WO 98/08849; WO 98/22461; WO 98/25929; WO98/38192; WO 99/01124; WO 99/02514; WO 99/03848; WO 99/07692; WO99/27890; and WO 99/28324; the disclosures of which are incorporatedherein by reference in their entirety.

Exemplary cryptophycin compounds are described in U.S. Pat. Nos.6,680,311 and 6,747,021.

Exemplary platinum compounds include cisplatin (PLATINOL®), carboplatin(PARAPLATIN®), oxaliplatin (ELOXATINE®), iproplatin, ormaplatin, andtetraplatin.

Still other classes of compounds or compounds with these or othercytotoxic modes of action may be selected, including, e.g., mitomycin C,mitomycin A, daunorubicin, doxorubicin, morpholino-doxorubicin,cyanomorpholino-doxorubicin, aminopterin, bleomycin,1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol,pyrrolobenzodiazepine (PBD) polyamide and dimers thereof. Other suitablecytotoxic agents include, for example, puromycins, topotecan, rhizoxin,echinomycin, combretastatin, netropsin, estramustine, cryptophysins,cemadotin, discodermolide, eleutherobin, and mitoxantrone.

Exemplary topoisomerase I inhibitors include camptothecin, camptothecinderivatives, camptothecin analogs and non-natural camptothecins, suchas, for example, CPT-11 (irinotecan), SN-38, GI-147211C, topotecan,9-aminocamptothecin, 7-hydroxymethyl camptothecin, 7-aminomethylcamptothecin, 10-hydroxycamptothecin, (20S)-camptothecin, rubitecan,gimatecan, karenitecin, silatecan, lurtotecan, exatecan, diflomotecan,belotecan, lurtotecan and S39625. Other camptothecin compounds that canbe used in the present disclosure include those described in, forexample, J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem., 23:554(1980); J. Med. Chem., 30:1774 (1987).

Angiogenesis inhibitors include, but are not limited, MetAP2 inhibitors,VEGF inhibitors, PIGF inhibitors, VGFR inhibitors, PDGFR inhibitors,MetAP2 inhibitors. Exemplary VGFR and PDGFR inhibitors include sorafenib(Nexavar), sunitinib (Sutent) and vatalanib. Exemplary MetAP2 inhibitorsinclude fumagillol analogs, meaning any compound that includes thefumagillin core structure, including fumagillamine, that inhibits theability of MetAP-2 to remove NH₂-terminal methionines from proteins asdescribed in Rodeschini et al., J. Org. Chem., 69, 357-373, 2004 andLiu, et al., Science 282, 1324-1327, 1998. Non limiting examples of“fumagillol analogs” are disclosed in J. Org. Chem., 69, 357, 2004; J.Org. Chem., 70, 6870, 2005; European Patent Application 0 354 787; J.Med. Chem., 49, 5645, 2006; Bioorg. Med. Chem., 11, 5051, 2003; Bioorg.Med. Chem., 14, 91, 2004; Tet. Lett. 40, 4797, 1999; WO99/61432; U.S.Pat. Nos. 6,603,812; 5,789,405; 5,767,293; 6,566,541; and 6,207,704.

Exemplary cell cycle progression inhibitors include CDK inhibitors suchas, for example, BMS-387032 and PD0332991; Rho-kinase inhibitors suchas, for example GSK429286; checkpoint kinase inhibitors such as, forexample, AZD7762; aurora kinase inhibitors such as, for example,AZD1152, MLN8054 and MLN8237; PLK inhibitors such as, for example, BI2536, BI6727 (Volasertib), GSK461364, ON-01910 (Estybon); and KSPinhibitors such as, for example, SB 743921, SB 715992 (ispinesib),MK-0731, AZD8477, AZ3146 and ARRY-520.

Exemplary PI3K/m-TOR/AKT signaling pathway inhibitors includephosphoinositide 3-kinase (PI3K) inhibitors, GSK-3 inhibitors, ATMinhibitors, DNA-PK inhibitors and PDK-1 inhibitors.

Exemplary PI3 kinase inhibitors are disclosed in U.S. Pat. No.6,608,053, and include BEZ235, BGT226, BKM120, CAL101, CAL263,demethoxyviridin, GDC-0941, GSK615, IC87114, LY294002, Palomid 529,perifosine, PI-103, PF-04691502, PX-866, SAR245408, SAR245409, SF1126,Wortmannin, XL147 and XL765.

Exemplary AKT inhibitors include, but are not limited to AT7867.

Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Rafand p38 MAPK inhibitors.

Exemplary MEK inhibitors are disclosed in U.S. Pat. No. 7,517,994 andinclude GDC-0973, GSK1120212, MSC1936369B, AS703026, RO5126766 andRO4987655, PD0325901, AZD6244, AZD 8330 and GDC-0973.

Exemplary B-raf inhibitors include CDC-0879, PLX-4032, and SB590885.

Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820 and SB202190.

Receptor tyrosine kinases (RTK) are cell surface receptors which areoften associated with signaling pathways stimulating uncontrolledproliferation of cancer cells and neoangiogenesis. Many RTKs, which overexpress or have mutations leading to constitutive activation of thereceptor, have been identified, including, but not limited to, VEGFR,EGFR, FGFR, PDGFR, EphR and RET receptor family receptors. Exemplaryspecific RTK targets include ErbB2, FLT-3, c-Kit, and c-Met.

Exemplary inhibitors of ErbB2 receptor (EGFR family) include but notlimited to AEE788 (NVP-AEE 788), BIBW2992, (Afatinib), Lapatinib,Erlotinib (Tarceva), and Gefitinib (Iressa).

Exemplary RTK inhibitors targeting more than one signaling pathway(multitargeted kinase inhibitors) include AP24534 (Ponatinib) thattargets FGFR, FLT-3, VEGFR-PDGFR and Bcr-Abl receptors; ABT-869(Linifanib) that targets FLT-3 and VEGFR-PDGFR receptors; AZD2171 thattargets VEGFR-PDGFR, Flt-1 and VEGF receptors; CHR-258 (Dovitinib) thattargets VEGFR-PDGFR, FGFR, Flt-3, and c-Kit receptors; Sunitinib(Sutent) that targets VEGFR, PDGFR, KIT, FLT-3 and CSF-IR; Sorafenib(Nexavar) and Vatalanib that target VEGFR, PDGFR as well asintracellular serine/threonine kinases in the Raf/Mek/Erk pathway.

Exemplary protein chaperon inhibitors include HSP90 inhibitors.Exemplary HSP90 inhibitors include 17AAG derivatives, BIIB021, BIIB028,SNX-5422, NVP-AUY-922 and KW-2478.

Exemplary HDAC inhibitors include Belinostat (PXD101), CUDC-101,Droxinostat, ITF2357 (Givinostat, Gavinostat), JNJ-26481585, LAQ824(NVP-LAQ824, Dacinostat), LBH-589 (Panobinostat), MC1568, MGCD0103(Mocetinostat), MS-275 (Entinostat), PCI-24781, Pyroxamide (NSC 696085),SB939, Trichostatin A and Vorinostat (SAHA).

Exemplary PARP inhibitors include iniparib (BSI 201), olaparib(AZD-2281), ABT-888 (Veliparib), AG014699, CEP 9722, MK 4827, KU-0059436(AZD2281), LT-673, 3-aminobenzamide, A-966492, and AZD2461.

Exemplary NAMPT inhibitors include FK866 (AP0866) and CHS828, GPP78,GMX1778 (CHS828), STF-118804, STF-31, CB 300919, CB 30865, GNE-617,IS001, TP201565, Nampt-IN-1, P7C3, WC-9528, CB30865, WI0479883 and(E)-N-(5-((4-(((2-(1H-Indol-3-yl)ethyl)(isopropyl)amino)methyl)phenyl)amino)pentyl)-3-(pyridin-3-yl)acrylamide.

Exemplary Wnt/Hedgehog signaling pathway inhibitors include vismodegib(RG3616/GDC-0449), cyclopamine (11-deoxojervine) (Hedgehog pathwayinhibitors) and XAV-939 (Wnt pathway inhibitor).

Exemplary RNA polymerase inhibitors include amatoxins. Exemplaryamatoxins include α-amanitins, β-amanitins, γ-amanitins, ε-amanitins,amanullin, amanullic acid, amaninamide, amanin, and proamanullin.

Exemplary protein synthesis inhibitors include trichothecene compounds.

In one embodiment, the drug is a topoisomerase inhibitor (such as, forexample, a non-natural camptothecin compound), vinca alkaloid, kinaseinhibitor (e.g., PI3 kinase inhibitor (GDC-0941 and PI-103)), MEKinhibitor, KSP inhibitor, RNA polymerase inhibitor, protein synthesisinhibitor, PARP inhibitor, NAMPT inhibitor, docetaxel, paclitaxel,doxorubicin, duocarmycin, auristatin, dolastatin, calicheamicins,topotecan, SN38, camptothecin, exatecan, nemorubicin and itsderivatives, PNU-159682, CC1065, elinafide, trichothecene,pyrrolobenzodiazepines, maytansinoids, DNA-binding drugs or a platinumcompound, and analogs thereof. In specific embodiments, the drug is aderivative of SN-38, camptothecin, topotecan, exatecan, calicheamicin,nemorubicin, PNU-159682, anthracycline, maytansinoid, taxane,trichothecene, CC1065, elinafide, vindesine, vinblastine, PI-103, AZD8330, dolastatin, auristatin E, auristatin F, a duocarmycin compound,ispinesib, pyrrolobenzodiazepine, ARRY-520 and stereoisomers, isosteresand analogs thereof.

In another embodiment, the drug used in the disclosure is a combinationof two or more drugs, such as, for example, PI3 kinase inhibitors andMEK inhibitors; broad spectrum cytotoxic compounds and platinumcompounds; PARP inhibitors, NAMPT inhibitors and platinum compounds;broad spectrum cytotoxic compounds and PARP inhibitors.

In yet another embodiment, the drug used in the disclosure is auristatinF-hydroxypropylamide-L-alanine.

In one embodiment, the Vinca alkaloid is a compound of Formula (V1),

wherein:

R₁₄ is hydrogen, —C(O)—C₁₋₃ alkyl or —C(O)-chloro substituted C₁₋₃alkyl;

R₁₅ is hydrogen, —CH₃ or —CHO;

when R₁₇ and R₁₈ are taken independently, R₁₈ is hydrogen, and eitherR₁₆ or R₁₇ is ethyl and the other is hydroxyl;

when R₁₇ and R₁₈ are taken together with the carbon to which they areattached to form an oxiran ring, R₁₆ is ethyl;

R₁₉ is hydrogen, OH, amino group, alkyl amino or —[C(R₂₀R₂₁)]_(a)—R₂₂;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(O CH₂—CH₂)_(f)—N(H)(R₂₃) or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is hydrogen or X² and NR₇₇ form a nitrogen containing heterocyclicmoiety;

R₈₂ is —NR₂₃ or oxygen;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3; and

f is an integer from 1 to 12.

Further examples of Vinca alkaloids are described in U.S. Pat. No.8,524,214B2 and US 2002/0103136.

In one embodiment the Vinca alkaloid of Formula (V1) is a compound ofFormula (VI1):

wherein:

R₄₀ is hydrogen, —OH, —NH₂, or any of the following structures:

wherein:

a is an integer from 1 to 6;

g is an integer from 2 to 6; and

c is an integer from 0 to 3.

In one embodiment, in Formula (VI1), R₄₀ is

In another embodiment, R₄₀ is

In another embodiment, R₄₀ is

In another embodiment, R₄₀ is

In another embodiment, R₄₀ is

In another embodiment, the compound of Formula (VI1) is a compound ofFormula (VIa), (VIb), (VIc), (VId), (VIe) or (VIf):

In another embodiment, the topoisomerase inhibitor is a camptothecincompound of Formula (VIII):

wherein:

R₂₄ is —H, —Cl, —F, —OH or alkyl; or R₂₄ and R₂₅, may be taken togetherto form an optionally substituted five- or six-membered ring;

R₂₅ is —H, —F, —OH, —CH₃, —CH═N—O-t-Butyl, —CH₂CH₂Si(CH₃)₃,—Si((CH₃)₂)-t-butyl, —O—C(O)—R₂₉;

R₂₉ is —NH₂, —R₂₈—C₁₋₆ alkyl-R₂₂, 5 to 12-membered heterocycloalkyl,R₂₈—C₅₋₁₂ heterocycloalkyl-C₁₋₆ alkyl-R₂₂ or —R₂₈—C₁₋₆ alkyl-C₆₋₁₂aryl-C₁₋₆ alkyl-R₂₂; or R₂₉ is R₄₇ as defined herein;

R₂₆ is —H, —CH₂—N(CH₃)₂, NH₂, or NO₂;

R₂₇ is —H, ethyl, N-methyl piperidine, cycloalkyl, —CH₂OH,—CH₂CH₂NHCH(CH₃)₂, or —N-4-methylcyclohexylamine;

R₇₉ is —H or —C(O)—R₂₈—[C(R₂₀R₂₁)]_(a)—R₂₂;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(O CH₂—CH₂)_(f)—N(H)(R₂₃), or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NR₂₃ or oxygen;

or R₂₆ and R₂₇ when taken together with the two carbon atoms to whichthey attach and the third carbon atom connecting the two carbon atomsform an optionally substituted six-membered ring;

R₂₈ is absent, NR₂₃ or oxygen;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3;

f is an integer from 1 to 12;

u is an integer 0 or 1;

w is an integer 0 or 1; and

with the proviso that the compound of Formula (VII1) must contain atleast one of R₂₉ and R₇₉.

In one embodiment the camptothecin compound of Formula (VII1) is acompound of Formula (VIII1), (VIIIa), or (VIIIb), or Formula (XXV) or(XXVa):

wherein:

R₃₀ is —NH₂, —R₂₈—[C(R₂₀R₂₁)]_(a)—R₂₂, —R₂₈—C₁₋₆ alkyl-R₂₂, 5 to12-membered heterocycloalkyl, R₂₈—C₅₋₁₂ heterocycloalkyl-C₁₋₆ alkyl-R₂₂or —R₂₈—C₁₋₆ alkyl-C₆₋₁₂ aryl-C₁₋₆ alkyl-R₂₂;

R₂₈ is absent, NR₂₃ or oxygen;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(O CH₂—CH₂)_(f)—N(H)(R₂₃) or—R₈₂—(C(O)—CH(X²)—NR₂₃)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NR₂₃ or oxygen;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3; and

f is an integer from 1 to 12.

In some embodiments, R₃₀ is any one of the following structures:

wherein:

a is an integer from 1 to 6;

c is an integer from 0 to 3; and

g is an integer from 2 to 6.

In one embodiment in Formula (VII1), R₃₀ is:

In another embodiment, the compound of Formula (VII1) is a compound ofFormula (VIIa), (VIIb), (VIIc), (VIId), (VIIe), (VIIf), (VIIg), (VIIh),(VIIi), or (VIIj):

In another embodiment the PI3 kinase inhibitor is a compound of Formula(IX1):

wherein

R₄₇ is an amino group, —R₉—[C(R₂₀R₂₁)]_(a)—R₁₀, —R₉—C₅₋₁₂heterocycloalkyl-C₁₋₆ alkyl-R₁₀, 5 to 12-membered heterocycloalkyl, or—R₉—C₆₋₁₀ aryl;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₁₀ is —OH, —NHR₈₃, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(O CH₂—CH₂)_(f)—N(H)(R₂₃),—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇ or —R₈₂—C(O)—[C(R₂₀R₂₁)]_(a)—R₈₂—R₈₃;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NR₂₃ or oxygen;

R₉ is absent, N—(R₈₃) or oxygen;

R₈₃ is hydrogen or CH₃; or

R₁₁:

each R₁₂ independently is hydrogen, chloride, —CH₃ or —OCH₃;

R₁₃ is hydrogen or —C(O)—(CH₂)_(d)—(O—CH₂—CH₂)_(f)—NH₂;

R₈₂ is —NR₂₃ or oxygen

X₄ is the side chain of lysine, arginine, citrulline, alanine orglycine;

X₅ is the side chain of phenylalanine, valine, leucine, isoleucine ortryptophan;

each of X₆ and X₇ is independently the side chain of glycine, alanine,serine, valine or proline;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3;

f is an integer from 1 to 12; and

each u independently is an integer 0 or 1;

or R₁₁ is —Y_(u)—W_(q)—R₈₈,

wherein:

Y is any one of the following structures:

in each of which the terminal NR₈₃ group of Y is proximal to R₈₈;

R₈₃ is hydrogen or CH₃,

each W is an amino acid unit;

each R₁₂′ independently is halogen, —C₁₋₈ alkyl, —O—C₁₋₈ alkyl, nitro orcyano;

R₈₈ is hydrogen or —C(O)—(CH₂)_(ff)—(NH—C(O))_(aa)-E_(j)-(CH₂)_(bb)—R₈₅

R₈₅ is NH₂ or OH;

E is —CH₂— or —CH₂CH₂O—;

u is an integer 0 or 1;

q is an integer from 0 to 12;

aa is an integer 0 or 1;

bb is an integer 0 or 2;

ff is an integer from 0 to 10;

h is an integer from 0 to 4;

j is an integer from 0 to 12; and

when E is —CH₂—, bb is 0 and j is an integer from 0 to 10; and when E is—CH₂CH₂—O—, bb is 2 and j is an integer from 1 to 12;

or R₁₁ is:

wherein:

R₈₃ is hydrogen or CH₃;

R₈₄ is C₁₋₆ alkyl or C₆₋₁₀ aryl;

each R₁₂′ independently is halogen, —C₁₋₈ alkyl, —O—C₁₋₈ alkyl, nitro orcyano;

h is an integer from 0 to 4; and

u is an integer 0 or 1.

In some embodiments, R₁₁ is:

wherein:

each R₁₂′ independently is chloride, —CH₃ or —OCH₃;

R₈₈ is hydrogen or —C(O)—(CH₂)_(ff)—(CH₂—CH₂O)_(j)—CH₂—CH₂—NH₂;

R₈₂ is —NR₂₃ or oxygen

X₄ is the side chain of lysine, arginine, citrulline, alanine orglycine;

X₅ is the side chain of phenylalanine, valine, leucine, isoleucine ortryptophan;

each of X₆ and X₇ is independently the side chain of glycine, alanine,serine, valine or proline;

ff is an integer from 1 to 3;

j is an integer from 1 to 12

h is an integer from 0 to 4; and

each u independently is an integer 0 or 1.

In some embodiments,

is citrulline-valine; lysine-phenylalanine; citrulline-phenylalanine;citrulline-leucine; citrulline-valine-glycine-glycine;glycine-phenylalanine-glycine-glycine; valine; proline; leucine orisoleucine.

In another embodiment, R₁₁ is any one of the following structures:

In some embodiments, R₄₇ is any one of the following structures:

wherein:

a is an integer from 1 to 6;

c is an integer from 0 to 3; and

g is an integer from 2 to 6.

In another embodiment the auristatin is a compound of Formula (X):

wherein:

each of R₃₁ and R₃₂ independently is hydrogen or C₁₋₈ alkyl and at mostone of R₃₁ and R₃₂ is hydrogen;

R₃₃ is hydrogen, C₁₋₈ alkyl, C₃₋₈ carbocycle, C₆₋₁₀ aryl, C₁₋₈alkyl-C₆₋₁₀ aryl, X¹—(C₃₋₈ carbocycle), C₃₋₈ heterocycle or X¹—(C₃₋₈heterocycle);

R₃₄ is hydrogen, C₁₋₈ alkyl, C₃₋₈ carbocycle, C₆₋₁₀ aryl, X¹—C₆₋₁₀ aryl,X¹—(C₃₋₈ carbocycle), C₃₋₈ heterocycle or X¹—(C₃₋₈ heterocycle);

R₃₅ is hydrogen or methyl;

or R₃₄ and R₃₅, together with the carbon atom to which they attach forma carbocyclic ring having the formula —(CR₅₅R₄₁)_(b)— wherein each ofR₅₅ and R₄₁ independently is hydrogen or C₁₋₈ alkyl and b is an integerfrom 3 to 7;

R₃₆ is hydrogen or C₁₋₈ alkyl;

R₃₇ is hydrogen, C₁₋₈ alkyl, C₃₋₈ carbocycle, C₆₋₁₀ aryl, —X¹—C₆₋₁₀aryl, —X¹—(C₃₋₈ carbocycle), C₃₋₈ heterocycle or —X¹—(C₃₋₈ heterocycle);

each R₃₈ independently is hydrogen, OH, C₁₋₈ alkyl, C₃₋₈ carbocycle orO—(C₁₋₈ alkyl);

R₅₃ is:

or R₅₄;

R₃₉ is H, C₁₋₈ alkyl, C₆₋₁₀ aryl, —X¹—C₆₋₁₀ aryl, C₃₋₈ carbocycle, C₃₋₈heterocycle, —X¹—C₃₋₈ heterocycle, —C₁₋₈ alkylene-NH₂, or (CH₂)₂SCH₃;

each X¹ independently is C₁₋₁₀ alkylene or C₃₋₁₀ cycloalkylene;

R₄₄ is hydrogen or C₁₋₈ alkyl;

R₄₅ is X³—R₄₂ or NH—R₁₉;

X³ is O or S;

R₁₉ is hydrogen, OH, amino group, alkyl amino or —[C(R₂₀R₂₁)]_(a)—R₂₂;

R₄₂ is an amino group, C₁₋₆ alkyl amino or —[C(R₂₀R₂₁)]_(a)—R₂₂;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₂₂ is —OH, —NHR₂₃, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(O CH₂—CH₂)_(f)—N(H)(R₂₃) or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NR₂₃ or oxygen;

R₅₄ is —C(R₅₆)₂—C(R₅₆)₂—C₆₋₁₀ aryl, —C(R₅₆)₂—C(R₅₆)₂—C₃₋₈ heterocycle or—C(R₅₆)₂—C(R₅₆)₂—C₃₋₈ carbocycle;

R₅₆ is independently selected from H, OH, C₁₋₈ alkyl, C₃₋₈ carbocycle,—O—C₁₋₈ alkyl, —O—C(O)—R₂₉ and —O—R₂₃—O—C₁₋₆ alkyl-NH₂;

R₂₉ is an amino group, 5 to 12-membered heterocycloalkyl, —R₂₈—C₁₋₆alkyl-R₂₂, R₂₈—C₅₋₁₂ heterocycloalkyl-C₁₋₆ alkyl-R₂₂,—[C(R₂₀R₂₁)]_(a)—R₂₂, or —R₂₈—C₁₋₆ alkyl-C₆₋₁₂ aryl-C₁₋₆ alkyl-R₂₂; orR₂₉ is R₄₇ as defined herein;

R₂₈ is absent, NR₂₃ or oxygen;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3; and

f is an integer from 1 to 12.

In some embodiments, in the auristatin compound of Formula (X):

R₃₉ is benzyl or

and

R₄₄ is hydrogen.

In another embodiment the auristatin is a compound of Formula (Xa):

wherein:

R₃₃ through R₃₈, and R₄₄ are as defined herein,

one of R₃₁ and R₃₂ is hydrogen or C₁₋₈ alkyl and the other is:

wherein:

R₈₃ is hydrogen or CH₃,

R₈₄ is C₁₋₆ alkyl or C₆₋₁₀ aryl;

each R₁₂′ independently is halogen, —C₁₋₈ alkyl, —O—C₁₋₈ alkyl, nitro orcyano;

h is an integer from 0 to 4; and

u is an integer 0 or 1;

R₅₃ is:

or R₅₄

R₃₉ is H, C₁₋₈ alkyl, C₆₋₁₀ aryl, —X¹—C₆₋₁₀ aryl, C₃₋₈ carbocycle, C₃₋₈heterocycle, —X¹—C₃₋₈ heterocycle, —C₁₋₈ alkylene-NH₂, or (CH₂)₂SCH₃,

each X¹ independently is C₁₋₁₀ alkylene or C₃₋₁₀ cycloalkylene;

R₄₅ is X³—R₄₂ or NH—R₁₉;

X³ is O or S;

R₁₉ is hydrogen, OH, amino group, alkyl amino or —[C(R₂₀R₂₁)]_(a)—R₂₂;

R₄₂ is H, an amino group, C₁₋₆ alkyl amino or —[C(R₂₀R₂₁)]_(a)—R₂₂;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₂₂ is —OH, —NHR₂₃, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(O—CH₂—CH₂)_(f)—N(H)(R₂₃) or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NR₂₃ or oxygen;

R₅₄ is —C(R₅₆)₂—C(R₅₆)₂—C₆₋₁₀ aryl, —C(R₅₆)₂—C(R₅₆)₂—C₃₋₈ heterocycle or—C(R₅₆)₂—C(R₅₆)₂—C₃₋₈ carbocycle;

R₅₆ is independently selected from H, OH, C₁₋₈ alkyl, C₃₋₈ carbocycle,—O—C₁₋₈ alkyl, —O—C(O)—R₂₉ and —O—R₂₃—O—C₁₋₆ alkyl-NH₂;

R₂₉ is an amino group, 5 to 12-membered heterocycloalkyl, —R₂₈—C₁₋₆alkyl-R₂₂, R₂₈—C₅₋₁₂ heterocycloalkyl-C₁₋₆ alkyl-R₂₂,—[C(R₂₀R₂₁)]_(a)—R₂₂, or —R₂₈—C₁₋₆ alkyl-C₆₋₁₂ aryl-C₁₋₆ alkyl-R₂₂; orR₂₉ is R₄₇ as defined herein;

R₂₈ is absent, NR₂₃ or oxygen;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3; and

f is an integer from 1 to 12.

In one embodiment, the auristatin compound of Formula (Xa) is a compoundof Formula (XIa) or Formula (XIb):

wherein:

R₉₂ is:

and

R₈₃ is hydrogen or CH₃.

In one embodiment the auristatin of Formula (X) is a compound of Formula(XI), Formula (XII) or Formula (XIII):

wherein the compound of Formula (XI) is:

wherein R₃₁ is H or CH₃ and R₄₂ is —CH₃ or any one of the followingstructures:

wherein:

a is an integer from 1 to 6;

c is an integer from 0 to 3; and

g is an integer from 2 to 6;

wherein the compound of Formula (XII) is:

wherein R₃₁ is H or CH₃ and R₄₀ is hydrogen, —OH, —NH₂, or any of thefollowing structures:

wherein:

a is an integer from 1 to 6;

g is an integer from 2 to 6; and

c is an integer from 0 to 3;

wherein the compound of Formula (XIII) is:

wherein:

R₃₁ is H or CH₃;

R₂₉ is an amino group, 5 to 12-membered heterocycloalkyl, —R₂₈—C₁₋₆alkyl-R₂₂, R₂₈—C₅₋₁₂ heterocycloalkyl-C₁₋₆ alkyl-R₂₂,—R₂₈—[C(R₂₀R₂₁)]_(a)—R₂₂, or —R₂₈—C₁₋₆ alkyl-C₆₋₁₂ aryl-C₁₋₆ alkyl-R₂₂;or R₂₉ is R₄₇ as defined herein;

-   -   each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀        aryl, hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to        12-membered heterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈        cycloalkyl, polyhydroxylated C₃₋₈ cycloalkyl or a side chain of        a natural or unnatural amino acid;    -   R₂₂ is —OH, —NHR₂₃, —COOH,        —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃), —R₈₂—C(O)(CH₂)_(d)—(O        CH₂—CH₂)_(f)—N(H)(R₂₃) or —R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NR₂₃ or oxygen;

R₂₈ is absent, NR₂₃ or oxygen;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3; and

f is an integer from 1 to 12.

In one embodiment, in Formula (XII), R₄₀ is

In another embodiment, the compound of Formula (XII) is a compound ofFormula (XIIa), (XIIb), (XIIc), (XIId), (XIIe), (XIIf), (XIIg) or(XIIh):

In one embodiment in the compound of Formula (XIII), R₂₉ is —NH₂, 5membered heterocycloalkyl, —R₂₈—C₁₋₆ alkyl-R₂₂, R₂₈—C₅₋₁₂heterocycloalkyl-C₁₋₆ alkyl-R₂₂ or —R₂₈—C₁₋₆ alkyl-C₆₋₁₂ aryl-C₁₋₆alkyl-R₂₂; or R₂₉ is R₄₇ as defined herein;

R₂₈ is absent, NR₂₃ or oxygen;

R₂₂ is —OH, —NHR₂₃, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(O CH₂—CH₂)_(f)—N(H)(R₂₃) or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NR₂₃ or oxygen;

c is an integer from 0 to 3;

d is an integer from 1 to 3; and

f is an integer from 1 to 12.

In yet another embodiment, R₂₉ is any one of the following structures:

wherein:

a is an integer from 1 to 6;

c is an integer from 0 to 3; and

g is an integer from 2 to 6.

In one embodiment, the MEK inhibitor is a compound of Formula (XIV):

wherein:

R₄₃ is H or —R₄₆—R₄₇;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(O CH₂—CH₂)_(f)—N(H)(R₂₃) or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NR₂₃ or oxygen;

R₄₆ is —C(O)—; —C(O)—O—, —C(O)—NH—, or absent;

R₄₇ is as defined herein;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3; and

f is an integer from 1 to 12.

Further examples of the MEK inhibitor are disclosed in U.S. Pat. No.7,517,994 B2.

In some embodiments, R₄₃ is —C(O)—(CH₂)_(a)—NH₂, or—C(O)—C(H)(CH₃)—(CH₂)_(c)—NH₂; in which a is an integer from 1 to 6; andc is an integer from 0 to 3.

In another embodiment, the duocarmycin compound is a compound of Formula(XV):

wherein:

R₄₇ is as defined herein;

R₄₈ is hydrogen, —COOC₁₋₆ alkyl, —COOH, —NH₂ or —CH₃;

R₄₉ is Cl, Br or —OH;

R₅₀ is hydrogen, —OCH₃,

each of R₅₁ and R₅₂ independently is hydrogen or —OCH₃; and

ring AA is either a phenyl or pyrrolyl ring.

Further examples of duocarmycin compounds are disclosed in U.S. Pat. No.7,553,816.

In one embodiment the duocarmycin compound of Formula (XV) is a compoundof Formula (XVI), (XVII), (XVIII) or (XIX):

wherein:

R₄₉ is Cl, Br or —OH; and

R₄₇ is as defined herein.

In another embodiment, the duocarmycin compound is a duocarmycin SAcompound of Formula (XX) or (XXI):

wherein:

R₄₂ is C₁₋₆ alkyl amino or —[C(R₂₀R₂₁)]_(a)—R₂₂;

each of R₂₀ and R₂₁ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl,hydroxylated C₆₋₁₀ aryl, polyhydroxylated C₆₋₁₀ aryl, 5 to 12-memberedheterocycle, C₃₋₈ cycloalkyl, hydroxylated C₃₋₈ cycloalkyl,polyhydroxylated C₃₋₈ cycloalkyl or a side chain of a natural orunnatural amino acid;

R₂₂ is —OH, —NH₂, —COOH, —R₈₂—C(O)(CH₂)_(c)—C(H)(R₂₃)—N(H)(R₂₃),—R₈₂—C(O)(CH₂)_(d)—(O CH₂—CH₂)_(f)—N(H)(R₂₃), or—R₈₂—(C(O)—CH(X²)—NH)_(d)—R₇₇;

each R₂₃ independently is hydrogen, C₁₋₆ alkyl, C₆₋₁₀ aryl, C₃₋₈cycloalkyl, —COOH, or —COO—C₁₋₆ alkyl;

X² is a side chain of a natural or unnatural amino acid;

R₇₇ is a hydrogen or X² and NR₇₇ form a nitrogen containing cycliccompound;

R₈₂ is —NR₂₃ or oxygen;

a is an integer from 1 to 6;

c is an integer from 0 to 3;

d is an integer from 1 to 3; and

f is an integer from 1 to 12.

In some embodiments, R₄₂ is any one of the following structures:

wherein:

a is an integer from 1 to 6;

g is an integer from 2 to 6; and

c is an integer from 0 to 3.

In another embodiment, the KSP inhibitor compound is a compound ofFormula (XXVI):

wherein R₃₀ is as defined herein.

In some embodiments, R₃₀ is:

wherein:

a is an integer from 1 to 6;

c is an integer from 0 to 3; and

g is an integer from 2 to 6.

In another embodiment, the duocarmycin compound is Duocarmycin A,Duocarmycin B1, Duocarmycin B2, Duocarmycin C1, Duocarmycin C2,Duocarmycin D, CC-1065, Adozelesin, Bizelesin or Carzelesin. Additionalduocarmycin compounds suitable for the conjugates, scaffolds and methodsof the disclosure are described in U.S. Pat. No. 5,101,038.

In another embodiment the KSP inhibitor compound is a compound ofFormula (XXVII), (XXVIII) or (XXIX):

wherein:

R₅₁ is a bond, —C(O)—(CH₂)—C(O)NH—(CH₂)₂—NH—,—C(O)—(CH₂O—CH₂)—C(O)NH—(CH₂)₂—NH—, or R₁₁ is as defined herein.

One skilled in the art of therapeutic agents will readily understandthat each of the therapeutic agents described herein can be modified insuch a manner that the resulting compound still retains the specificityand/or activity of the original compound. The skilled artisan will alsounderstand that many of these compounds can be used in place of thetherapeutic agents described herein. Thus, the therapeutic agentsdisclosed herein include analogs and derivatives of the compoundsdescribed herein.

Table A below provides more examples of the therapeutic agents andderivatives thereof suitable for conjugation to form the antibody-drugconjugates or drug-carrying scaffolds of the disclosure. Spectral dataof certain compounds are also provided (ND in the table means “notdetermined”). These examples may also be the active form of the drugwhen it is released from the conjugates in vitro or in vivo.

TABLE A (VI1)

R₄₀

(IX1)

R₄₇ m/z

ND

ND

ND

ND (XI)

R₄₂ m/z H —CH₃ 760

802.6

790

804 (XII)

R₄₀ m/z —H

803.5

789.1

974.2

874.5

902.2

ND

ND —OH 788

803.4

803.4

874.4

874.4

874.4

874.4

900.2

900.2

900.5

900.5

1016.6

989.5

975.5 (XIII)

—C(O)—R₂₉ m/z

903.2

803.1

790

832.6

829.1

802 (XIV)

R₄₃ m/z

ND

644.9 (XVII)

R₄₇ m/z

553.1

538.1

654.1

566.1

568.1

ND

ND

667.2

622.2

632.02

986.2

ND

ND (XXVII)

(XXVIII)

(XXIX)

m/z R₁₁ (XXVII)

922.3

732.2

ND

ND

ND

ND

Hydrophilic Group or T¹

In one embodiment, the hydrophilic group included in the conjugates orscaffolds of the disclosure is a water-soluble and substantiallynon-antigenic polymer. Examples of the hydrophilic group, include, butare not limited to, polyalcohols, polyethers, polyanions, polycations,polyphosphoric acids, polyamines, polysaccharides, polyhydroxycompounds, polylysines, and derivatives thereof. One end of thehydrophilic group can be functionalized so that it can be covalentlyattached to the Multifunctional Linker or M^(A) linker (e.g., to anamino acid in the M^(A) linker) by means of a non-cleavable linkage orvia a cleavable linkage. Functionalization can be, for example, via anamine, thiol, NHS ester, maleimide, alkyne, azide, carbonyl, or otherfunctional group. The other terminus (or termini) of the hydrophilicgroup will be free and untethered. By “untethered”, it is meant that thehydrophilic group will not be attached to another moiety, such as D or aDrug Unit, Releasable Assembly Unit, or other components of theconjugates or scaffolds of the disclosure. The free and untethered endof the hydrophilic group may include a methoxy, carboxylic acid, alcoholor other suitable functional group. The methoxy, carboxylic acid,alcohol or other suitable functional group acts as a cap for theterminus or termini of the hydrophilic group.

A cleavable linkage refers to a linkage that is not substantiallysensitive to cleavage while circulating in the plasma but is sensitiveto cleavage in an intracellular or intratumoral environment. Anon-cleavable linkage is one that is not substantially sensitive tocleavage in any biological environment. Chemical hydrolysis of ahydrazone, reduction of a disulfide, and enzymatic cleavage of a peptidebond or glycosidic linkage are examples of cleavable linkages. Exemplaryattachments of the hydrophilic group are via amide linkages, etherlinkages, ester linkages, hydrazone linkages, oxime linkages, disulfidelinkages, peptide linkages or triazole linkages. In some embodiments,the attachment of the hydrophilic group to the Multifunctional Linker orM^(A) linker (e.g., to an amino acid in the M^(A) linker) is via anamide linkage.

For those embodiments wherein the conjugate or scaffold of thedisclosure comprises more than one hydrophilic groups, the multiplehydrophilic groups may be the same or different chemical moieties (e.g.,hydrophilic groups of different molecular weight, number of subunits, orchemical structure). The multiple hydrophilic groups can be attached tothe Multifunctional Linker or M^(A) linker at a single attachment siteor different sites.

The addition of the hydrophilic group may have two potential impactsupon the pharmacokinetics of the resulting conjugate. The desired impactis the decrease in clearance (and consequent in increase in exposure)that arises from the reduction in non-specific interactions induced bythe exposed hydrophobic elements of the drug or drug-linker. The secondimpact is undesired impact and is the decrease in volume and rate ofdistribution that may arise from the increase in the molecular weight ofthe conjugate. Increasing the molecular weight of the hydrophilic groupincreases the hydrodynamic radius of a conjugate, resulting in decreaseddiffusivity that may diminish the ability of the conjugate to penetrateinto a tumor. Because of these two competing pharmacokinetic effects, itis desirable to use a hydrophilic group that is sufficiently large todecrease the conjugate clearance thus increasing plasma exposure, butnot so large as to greatly diminish its diffusivity, which may reducethe ability of the conjugate to reach the intended target cellpopulation.

In some embodiments, the hydrophilic group, includes, but is not limitedto, a sugar alcohol (also known as polyalcohol, polyhydric alcohol,alditol or glycitol, such as inositol, glycerol, erythritol, threitol,arabitol, xylitol, ribitol, galactitol, mannitol, sorbitol, and thelike) or a derivative thereof (e.g., amino polyalcohol), carbohydrate(e.g., a saccharide), a polyvinyl alcohol, a carbohydrate-based polymer(e.g., dextrans), a hydroxypropylmethacrylamide (HPMA), a polyalkyleneoxide, and/or a copolymer thereof.

In one embodiment, the hydrophilic group comprises a plurality ofhydroxyl (“—OH”) groups, such as moieties that incorporatemonosaccharides, oligosaccharides, polysaccharides, and the like. In yetanother embodiment the hydrophilic group comprises a plurality of—(CR₅₈OH)— groups, wherein R₅₈ is hydrogen or C₁₋₈ alkyl.

In some embodiments, the hydrophilic group comprises one or more of thefollowing fragments of the formula:

in which

n₁ is an integer from 0 to about 6;

each R₅₈ is independently hydrogen or C₁₋₈ alkyl;

R₆₀ is a bond, a C₁₋₆ alkyl linker, or —CHR₅₉— in which R₅₉ is H, alkyl,cycloalkyl, or arylalkyl;

R₆₁ is CH₂OR₆₂, COOR₆₂, —(CH₂)_(n2)COOR₆₂, or a heterocycloalkylsubstituted with one or more hydroxyl;

R₆₂ is H or C₁₋₈ alkyl; and

n₂ is an integer from 1 to about 5.

For example, R₅₈ is hydrogen, R₆₀ is a bond or a C₁₋₆ alkyl linker, n₁is an integer from 1 to about 6, and R₆₁ is CH₂OH or COOH. For example,R₅₈ is hydrogen, R₆₀ is —CHR₅₉—, n₁ is 0, and R₆₁ is a heterocycloalkylsubstituted with one or more hydroxyl, e.g., a monosaccharide.

In some embodiments, the hydrophilic group comprises a glucosyl-amine, adi-amine or a tri-amine.

In some embodiments, the hydrophilic group comprises one or more of thefollowing fragments or a stereoisomer thereof:

wherein:

R₅₉ is H, alkyl, cycloalkyl, or arylalkyl;

n₁ is an integer from 1 to about 6;

n₂ is an integer from 1 to about 5; and

n₃ is an integer from about 1 to about 3.

It is understood that all stereochemical forms of the hydrophilic groupsare contemplated herein. For example, in the above formula, thehydrophilic group may be derived from ribose, xylose, glucose, mannose,galactose, or other sugar and retain the stereochemical arrangements ofpendant hydroxyl and alkyl groups present on those molecules. Inaddition, it is to be understood that in the foregoing formulae, variousdeoxy compounds are also contemplated. Illustratively, one or more ofthe following features are contemplated for the hydrophilic groups whenapplicable:

For example, n₃ is 2 or 3.

For example, n₁ is 1, 2, or 3.

For example, n₂ is 1.

For example, R₅₉ is hydrogen.

For example, the hydrophilic group comprises:

For example, the hydrophilic group comprises:

For example, the hydrophilic group comprises:

In some embodiments, the hydrophilic group comprises

in which

n₄ is an integer from 1 to about 25;

each R₆₃ is independently hydrogen or C₁₋₈ alkyl;

R₆₄ is a bond or a C₁₋₈ alkyl linker;

R₆₅ is H, C₁₋₈ alkyl, or —(CH₂)_(n2)COOR₆₂;

R₆₂ is H or C₁₋₈ alkyl; and

n₂ is an integer from 1 to about 5.

In some embodiments, the hydrophilic group comprises:

For example, n₄ is an integer from about 2 to about 20, from about 4 toabout 16, from about 6 to about 12, from about 8 to about 12.

For example, n₄ is 6, 7, 8, 9, 10, 11, or 12.

In other embodiments, the hydrophilic group comprises a polyether, e.g.,a polyalkylene glycol (PAO). PAO includes but is not limited to,polymers of lower alkylene oxides, in particular polymers of ethyleneoxide, such as, for example, propylene oxide, polypropylene glycols,polyethylene glycol (PEG), polyoxyethylenated polyols, copolymersthereof and block copolymers thereof. In other embodiments thepolyalkylene glycol is a polyethylene glycol (PEG) including, but notlimited to, polydisperse PEG, monodisperse PEG and discrete PEG.Polydisperse PEGs are a heterogeneous mixture of sizes and molecularweights whereas monodisperse PEGs are typically purified fromheterogeneous mixtures and are therefore provide a single chain lengthand molecular weight. In another embodiment, the PEG units are discretePEGs provide a single molecule with defined and specified chain length.In some embodiments, the polyethylene glycol is mPEG.

In some embodiments, the hydrophilic group comprises a PEG unit whichcomprises one or multiple polyethylene glycol chains. The polyethyleneglycol chains can be linked together, for example, in a linear, branchedor star shaped configuration. The PEG unit, in addition to comprisingrepeating polyethylene glycol subunits, may also contain non-PEGmaterial (e.g., to facilitate coupling of multiple PEG chains to eachother or to facilitate coupling to the amino acid). Non-PEG materialrefers to the atoms in the PEG chain that are not part of the repeating—CH₂CH₂O— subunits. In one embodiment, the PEG chain can comprise twomonomeric PEG chains linked to each other via non-PEG elements. Inanother embodiment, the PEG Unit can comprise two linear PEG chainsattached to a central core that is attached to the amino acid (i.e., thePEG unit itself is branched).

The PEG unit may be covalently bound to the Multifunctional Linker orM^(A) linker (e.g., to an amino acid in the M^(A) linker) via a reactivegroup. Reactive groups are those to which an activated PEG molecule maybe bound (e.g., a free amino or carboxyl group). For example, N-terminalamino acids and lysines (K) have a free amino group; and C-terminalamino acid residues have a free carboxyl group. Sulfhydryl groups (e.g.,as found on cysteine residues) may also be used as a reactive group forattaching PEG.

In some embodiments, the PEG unit may be attached to the MultifunctionalLinker or M^(A) linker (e.g., to an amino acid in the M^(A) linker) byusing methoxylated PEG (“mPEG”) having different reactive moieties,including, but not limited to, succinimidyl succinate (SS), succinimidylcarbonate (SC), mPEG-imidate, para-nitrophenylcarbonate (NPC),succinimidyl propionate (SPA), and cyanuric chloride. Examples of mPEGsinclude, but are not limited to, mPEG-succinimidyl succinate (mPEG-SS),mPEG2-succinimidyl succinate (mPEG₂-SS), mPEG-succinimidyl carbonate(mPEG-SC), mPEG₂-succinimidyl carbonate (mPEG₂-SC), mPEG-imidate,mPEG-para-nitrophenylcarbonate (mPEG-NPC), mPEG-imidate,mPEG₂-para-nitrophenylcarbonate (mPEG₂-NPC), mPEG-succinimidylpropionate (mPEG-SPA), mPEG₂-succinimidyl propionate (mPEG₂-SPA),mPEG-N-hydroxy-succinimide (mPEG-NHS), mPEG₂-N-hydroxy-succinimide(mPEG₂-NHS), mPEG-cyanuric chloride, mPEG₂-cyanuric chloride,mPEG₂-Lysinol-NPC, and mPEG₂-Lys-NHS. A wide variety of PEG species canbe used, and substantially any suitable reactive PEG reagent can beused. In some embodiments, the reactive PEG reagent will result information of a carbamate or amide bond upon attachment to theMultifunctional Linker or M^(A) linker (e.g., to an amino acid in theM^(A) linker). The reactive PEG reagents include, but are not limitedto, mPEG₂-N-hydroxy-succinimide (mPEG₂-NHS), bifunctional PEGpropionaldehyde (mPEG₂-ALD), multi-Arm PEG, maleimide-containing PEG(mPEG(MAL)₂, mPEG₂(MAL)), mPEG-NH₂, mPEG-succinimidyl propionate(mPEG-SPA), succinimide of mPEG butanoate acid (mPEG-SBA),mPEG-thioesters, mPEG-Double Esters, mPEG-BTC, mPEG-ButyrALD,mPEG-acetaldehyde diethyl acetal (mPEG-ACET), heterofunctional PEGs(e.g., NH₂-PEG-COOH, Boc-PEG-NHS, Fmoc-PEG-NHS, NHS-PEG-vinylsulfone(NHS-PEG-VS), or NHS-PEG-MAL), PEG acrylates (ACRL-PEG-NHS),PEG-phospholipids (e.g., mPEG-DSPE), multi-armed PEGs of the SUNBRITE™series including the glycerine-based PEGs activated by a chemistrychosen by those skilled in the art, any SUNBRITE activated PEGs(including but not limited to carboxyl-PEGs, p-NP-PEGs, Tresyl-PEGs,aldehyde PEGs, acetal-PEGs, amino-PEGs, thiol-PEGs, maleimido-PEGs,hydroxyl-PEG-amine, amino-PEG-COOK hydroxyl-PEG-aldehyde, carboxylicanhydride type-PEG, functionalized PEG-phospholipid, and other similarand/or suitable reactive PEGs.

In some embodiments, the PEG unit comprises at least 6 subunits, atleast 7 subunits, at least 8 subunits, at least 9 subunits, at least 10subunits, at least 11 subunits, at least 12 subunits, at least 13subunits, at least 14 subunits, at least 15 subunits, at least 16subunits, at least 17 subunits, at least 18 subunits, at least 19subunits, at least 20 subunits, at least 21 subunits, at least 22subunits, at least 23 subunits, or at least 24 subunits. In some suchembodiments, the PEG unit comprises no more than about 72 subunits.

In some embodiments, the PEG unit comprises at least 6 subunits, atleast 7 subunits, at least 8 subunits, at least 9 subunits, at least 10subunits, at least 11 subunits, at least 12 subunits, at least 13subunits, at least 14 subunits, at least 15 subunits, at least 16subunits, at least 17 subunits, at least 18 subunits, at least 19subunits, at least 20 subunits, at least 21 subunits, at least 22subunits, at least 23 subunits, or at least 24 subunits.

In some embodiments, the PEG unit comprises at least 6 subunits, atleast 7 subunits, at least 8 subunits, at least 9 subunits, at least 10subunits, at least 11 subunits, at least 12 subunits, at least 13subunits, at least 14 subunits, at least 15 subunits, at least 16subunits, at least 17 subunits, or at least 18 subunits.

In some embodiments, the PEG unit comprises at least 6 subunits, atleast 7 subunits, at least 8 subunits, at least 9 subunits, at least 10subunits, at least 11 subunits, or at least 12 subunits.

In some embodiments, the PEG unit comprises at least 8 subunits, atleast 9 subunits, at least 10 subunits, at least 11 subunits, or atleast 12 subunits.

In some embodiments, the PEG unit comprises at least 6 subunits, atleast 7 subunits, or at least 8 subunits.

In some embodiments, the PEG unit comprises one or more linear PEGchains each having at least 2 subunits, at least 3 subunits, at least 4subunits, at least 5 subunits, at least 6 subunits, at least 7 subunits,at least 8 subunits, at least 9 subunits, at least 10 subunits, at least11 subunits, at least 12 subunits, at least 13 subunits, at least 14subunits, at least 15 subunits, at least 16 subunits, at least 17subunits, at least 18 subunits, at least 19 subunits, at least 20subunits, at least 21 subunits, at least 22 subunits, at least 23subunits, or at least 24 subunits. In another embodiment, the PEG unitcomprises a combined total of at least 6 subunits, at least 8, at least10 subunits, or at least 12 subunits. In some such embodiments, the PEGunit comprises no more than a combined total of about 72 subunits,preferably no more than a combined total of about 36 subunits.

In some embodiments, the PEG unit comprises a combined total of from 4to 72, 4 to 60, 4 to 48, 4 to 36 or 4 to 24 subunits, from 5 to 72, 5 to60, 5 to 48, 5 to 36 or 5 to 24 subunits, from 6 to 72, 6 to 60, 6 to48, 6 to 36 or from 6 to 24 subunits, from 7 to 72, 7 to 60, 7 to 48, 7to 36 or 7 to 24 subunits, from 8 to 72, 8 to 60, 8 to 48, 8 to 36 or 8to 24 subunits, from 9 to 72, 9 to 60, 9 to 48, 9 to 36 or 9 to 24subunits, from 10 to 72, 10 to 60, 10 to 48, 10 to 36 or 10 to 24subunits, from 11 to 72, 11 to 60, 11 to 48, 11 to 36 or 11 to 24subunits, from 12 to 72, 12 to 60, 12 to 48, 12 to 36 or 12 to 24subunits, from 13 to 72, 13 to 60, 13 to 48, 13 to 36 or 13 to 24subunits, from 14 to 72, 14 to 60, 14 to 48, 14 to 36 or 14 to 24subunits, from 15 to 72, 15 to 60, 15 to 48, 15 to 36 or 15 to 24subunits, from 16 to 72, 16 to 60, 16 to 48, 16 to 36 or 16 to 24subunits, from 17 to 72, 17 to 60, 17 to 48, 17 to 36 or 17 to 24subunits, from 18 to 72, 18 to 60, 18 to 48, 18 to 36 or 18 to 24subunits, from 19 to 72, 19 to 60, 19 to 48, 19 to 36 or 19 to 24subunits, from 20 to 72, 20 to 60, 20 to 48, 20 to 36 or 20 to 24subunits, from 21 to 72, 21 to 60, 21 to 48, 21 to 36 or 21 to 24subunits, from 22 to 72, 22 to 60, 22 to 48, 22 to 36 or 22 to 24subunits, from 23 to 72, 23 to 60, 23 to 48, 23 to 36 or 23 to 24subunits, or from 24 to 72, 24 to 60, 24 to 48, 24 to 36 or 24 subunits.

In some embodiments, the PEG unit comprises one or more linear PEGchains having a combined total of from 4 to 72, 4 to 60, 4 to 48, 4 to36 or 4 to 24 subunits, from 5 to 72, 5 to 60, 5 to 48, 5 to 36 or 5 to24 subunits, from 6 to 72, 6 to 60, 6 to 48, 6 to 36 or 6 to 24subunits, from 7 to 72, 7 to 60, 7 to 48, 7 to 36 or 7 to 24 subunits,from 8 to 72, 8 to 60, 8 to 48, 8 to 36 or 8 to 24 subunits, from 9 to72, 9 to 60, 9 to 48, 9 to 36 or 9 to 24 subunits, from 10 to 72, 10 to60, 10 to 48, 10 to 36 or 10 to 24 subunits, from 11 to 72, 11 to 60, 11to 48, 11 to 36 or 11 to 24 subunits, from 12 to 72, 12 to 60, 12 to 48,12 to 36 or 12 to 24 subunits, from 13 to 72, 13 to 60, 13 to 48, 13 to36 or 13 to 24 subunits, from 14 to 72, 14 to 60, 14 to 48, 14 to 36 or14 to 24 subunits, from 15 to 72, 15 to 60, 15 to 48, 15 to 36 or 15 to24 subunits, from 16 to 72, 16 to 60, 16 to 48, 16 to 36 or 16 to 24subunits, from 17 to 72, 17 to 60, 17 to 48, 17 to 36 or 17 to 24subunits, from 18 to 72, 18 to 60, 18 to 48, 18 to 36 or 18 to 24subunits, from 19 to 72, 19 to 60, 19 to 48, 19 to 36 or 19 to 24subunits, from 20 to 72, 20 to 60, 20 to 48, 20 to 36 or 20 to 24subunits, from 21 to 72, 21 to 60, 21 to 48, 21 to 36 or 21 to 24subunits, from 22 to 72, 22 to 60, 22 to 48, 22 to 36 or 22 to 24subunits, from 23 to 72, 23 to 60, 23 to 48, 23 to 36 or 23 to 24subunits, or from 24 to 72, 24 to 60, 24 to 48, 24 to 36 or 24 subunits.

In some embodiments, the PEG unit is a derivatized linear single PEGchain having at least 2 subunits, at least 3 subunits, at least 4subunits, at least 5 subunits, at least 6 subunits, at least 7 subunits,at least 8 subunits, at least 9 subunits, at least 10 subunits, at least11 subunits, at least 12 subunits, at least 13 subunits, at least 14subunits, at least 15 subunits, at least 16 subunits, at least 17subunits, at least 18 subunits, at least 19 subunits, at least 20subunits, at least 21 subunits, at least 22 subunits, at least 23subunits, or at least 24 subunits.

In some embodiments, the PEG unit is a derivatized linear single PEGchain having from 6 to 72, 6 to 60, 6 to 48, 6 to 36 or 6 to 24subunits, from 7 to 72, 7 to 60, 7 to 48, 7 to 36 or 7 to 24 subunits,from 8 to 72, 8 to 60, 8 to 48, 8 to 36 or 8 to 24 subunits, from 9 to72, 9 to 60, 9 to 48, 9 to 36 or 9 to 24 subunits, from 10 to 72, 10 to60, 10 to 48, 10 to 36 or 10 to 24 subunits, from 11 to 72, 11 to 60, 11to 48, 11 to 36 or 11 to 24 subunits, from 12 to 72, 12 to 60, 12 to 48,12 to 36 or 12 to 24 subunits, from 13 to 72, 13 to 60, 13 to 48, 13 to36 or 13 to 24 subunits, from 14 to 72, 14 to 60, 14 to 48, 14 to 36 or14 to 24 subunits, from 15 to 72, 15 to 60, 15 to 48, 15 to 36 or 15 to24 subunits, from 16 to 72, 16 to 60, 16 to 48, 16 to 36 or 16 to 24subunits, from 17 to 72, 17 to 60, 17 to 48, 17 to 36 or 17 to 24subunits, from 18 to 72, 18 to 60, 18 to 48, 18 to 36 or 18 to 24subunits, from 19 to 72, 19 to 60, 19 to 48, 19 to 36 or 19 to 24subunits, from 20 to 72, 20 to 60, 20 to 48, 20 to 36 or 20 to 24subunits, from 21 to 72, 21 to 60, 21 to 48, 21 to 36 or 21 to 24subunits, from 22 to 72, 22 to 60, 22 to 48, 22 to 36 or 22 to 24subunits, from 23 to 72, 23 to 60, 23 to 48, 23 to 36 or 23 to 24subunits, or from 24 to 72, 24 to 60, 24 to 48, 24 to 36 or 24 subunits.

In some embodiments, the PEG unit is a derivatized linear single PEGchain having from 2 to 72, 2 to 60, 2 to 48, 2 to 36 or 2 to 24subunits, from 2 to 72, 2 to 60, 2 to 48, 2 to 36 or 2 to 24 subunits,from 3 to 72, 3 to 60, 3 to 48, 3 to 36 or 3 to 24 subunits, from 3 to72, 3 to 60, 3 to 48, 3 to 36 or 3 to 24 subunits, from 4 to 72, 4 to60, 4 to 48, 4 to 36 or 4 to 24 subunits, from 5 to 72, 5 to 60, 5 to48, 5 to 36 or 5 to 24 subunits.

For example, a linear PEG unit is:

wherein;

the wavy line indicates site of attachment to the Multifunctional Linkeror M^(A) linker (e.g., to an amino acid in the M^(A) linker);

Y₇₁ is a PEG attachment unit;

Y₇₂ is a PEG capping unit;

Y₇₃ is an PEG coupling unit (i.e., for coupling multiple PEG subunitchains together);

d₉ is an integer from 2 to 72, preferably from 4 to 72, more preferablyfrom 6 to 72, from 8 to 72, from 10 to 72, from 12 to 72 or from 6 to24;

each d₁₀ is independently an integer from 1 to 72.

d₁₁ is an integer from 2 to 5.

In some embodiments, there are at least 6, preferably at least 8, atleast 10, or at least 12 PEG subunits in the PEG unit. In someembodiments, there are no more than 72 or 36 PEG subunits in the PEGunit.

In some embodiments, d₉ is 8 or about 8, 12 or about 12, 24 or about 24.

In some embodiments, each Y₇₂ is independently —C₁₋₁₀ alkyl, —C₂₋₁₀alkyl-CO₂H, —C₂₋₁₀ alkyl-OH, —C₂₋₁₀ alkyl-NH₂, —C₂₋₁₀ alkyl-NH(C₁₋₃alkyl), or C₂₋₁₀ alkyl-N(C₁₋₃ alkyl)₂.

In some embodiments, Y₇₂ is —C₁₋₁₀ alkyl, —C₂₋₁₀ alkyl-CO₂H, —C₂₋₁₀alkyl-OH, or —C₂₋₁₀ alkyl-NH₂.

The PEG coupling unit is part of the PEG unit and is non-PEG materialthat acts to connect two or more chains of repeating CH₂CH₂O— subunits.In some embodiments, the PEG coupling unit Y₇₃ is —C₂₋₁₀ alkyl-C(O)—NH—,alkyl-NH—C(O)—, —C₂₋₁₀ alkyl-NH—, —C₂₋₁₀ alkyl-C(O)—, —C₂₋₁₀ alkyl-O— or—C₂₋₁₀ alkyl-S—.

In some embodiments, each Y₇₃ is independently —C₁₋₁₀ alkyl-C(O)—NH—,—C₁₋₁₀ alkyl-NH—C(O)—, —C₂₋₁₀ alkyl-NH—, —C₂₋₁₀ alkyl-O—, —C₁₋₁₀alkyl-S—, or -alkyl-NH—.

The PEG attachment unit is part of the PEG unit and acts to link the PEGunit to the Multifunctional Linker or M^(A) linker (e.g., to an aminoacid in the M^(A) linker). For example, the amino acid has a functionalgroup that forms a bond with the PEG Unit. Functional groups forattachment of the PEG unit to the amino acid include sulfhydryl groupsto form disulfide bonds or thioether bonds, aldehyde, ketone, orhydrazine groups to form hydrazone bonds, hydroxylamine to form oximebonds, carboxylic or amino groups to form peptide bonds, carboxylic orhydroxy groups to form ester bonds, sulfonic acids to form sulfonamidebonds, alcohols to form carbamate bonds, and amines to form sulfonamidebonds or carbamate bonds or amide bonds. Accordingly, the PEG unit canbe attached to the amino acid, for example, via a disulfide, thioether,hydrazone, oxime, peptide, ester, sulfonamide, carbamate, or amide bond.Typically, the reaction for attaching the PEG unit can be acycloaddition, addition, addition/elimination or substitution reaction,or a combination thereof when applicable.

In some embodiments, the PEG attachment unit Y₇₁ is a bond, —C(O)—, —O—,—S—, —S(O)—, —S(O)₂—, —NR₅—, —C(O)O—, —C(O)—C₁₋₁₀ alkyl, —C(O)—C₁₋₁₀alkyl-O—, —C(O)—C₁₋₁₀ alkyl-CO₂—, —C(O)—C₁₋₁₀ alkyl-NR₅—, —C(O)—C₁₋₁₀alkyl-S—, —C(O)—C₁₋₁₀ alkyl-C(O)—NR₅—, —C(O)—C₁₋₁₀ alkyl-NR₅—C(O)—,—C₁₋₁₀ alkyl, —C₁₋₁₀ alkyl-O—, —C₁₋₁₀ alkyl-CO₂—, —C₁₋₁₀ alkyl-NR₅—,—C₁₋₁₀ alkyl-S—, —C₁₋₁₀ alkyl-C(O)—NR₅—, —C₁₋₁₀ alkyl-NR₅—C(O)—,—CH₂CH₂SO₂—C₁₋₁₀ alkyl-, —CH₂C(O)—C₁₋₁₀ alkyl-, ═N—(O or N)—C₁₋₁₀alkyl-O—, ═N—(O or N)—C₁₋₁₀ alkyl-NR₅—, ═N—(O or N)—C₁₋₁₀ alkyl-CO₂—,═N—(O or N)—C₁₋₁₀ alkyl-S—,

In some embodiments, Y₇₁ is —NH—, —C(O)—, a triazole group, —S—, or amaleimido-group such as

wherein the wavy line indicates attachment to the Multifunctional Linkeror M^(A) linker (e.g., to an amino acid in the M^(A) linker) and theasterisk indicates the site of attachment within the PEG Unit.

Examples of linear PEG units include:

wherein the wavy line indicates site of attachment to theMultifunctional Linker or M^(A) linker (e.g., to an amino acid in theM^(A) linker), and each d₉ is independently an integer from 4 to 24, 6to 24, 8 to 24, 10 to 24, 12 to 24, 14 to 24, or 16 to 24.

In some embodiments, d₉ is about 8, about 12, or about 24.

In other embodiments, the PEG unit is from about 300 daltons to about 5kilodaltons; from about 300 daltons, to about 4 kilodaltons; from about300 daltons, to about 3 kilodaltons; from about 300 daltons, to about 2kilodaltons; or from about 300 daltons, to about 1 kilodalton. In somesuch aspects, the PEG unit has at least 6 subunits or at least 8, 10 or12 subunits. In some embodiments, the PEG unit has at least 6 subunitsor at least 8, 10 or 12 subunits but no more than 72 subunits,preferably no more than 36 subunits.

Suitable polyethylene glycols may have a free hydroxy group at each endof the polymer molecule, or may have one hydroxy group etherified with alower alkyl, e.g., a methyl group. Also suitable for the practice of thedisclosure are derivatives of polyethylene glycols having esterifiablecarboxy groups. Polyethylene glycols are commercially available underthe trade name PEG, usually as mixtures of polymers characterized by anaverage molecular weight. Polyethylene glycols having an averagemolecular weight from about 300 to about 5000 are preferred, thosehaving an average molecular weight from about 600 to about 1000 beingparticularly preferred.

Other examples of hydrophilic groups that are suitable for theconjugates, scaffolds, and methods disclosed herein can be found ine.g., U.S. Pat. No. 8,367,065 column 13; U.S. Pat. No. 8,524,696 column6; WO2015/057699 and WO 2014/062697, the contents of each of which arehereby incorporated by reference in their entireties.

Protein-Based Recognition Molecules (PBRMs)

The protein-based recognition molecule directs the conjugates comprisinga peptide linker to specific tissues, cells, or locations in a cell. Theprotein-based recognition molecule can direct the conjugate in cultureor in a whole organism, or both. In each case, the protein-basedrecognition molecule has a ligand that is present on the cell surface ofthe targeted cell(s) to which it binds with an effective specificity,affinity and avidity. In some embodiments, the protein-based recognitionmolecule targets the conjugate to tissues other than the liver. In otherembodiments the protein-based recognition molecule targets the conjugateto a specific tissue such as the liver, kidney, lung or pancreas. Theprotein-based recognition molecule can target the conjugate to a targetcell such as a cancer cell, such as a receptor expressed on a cell suchas a cancer cell, a matrix tissue, or a protein associated with cancersuch as tumor antigen. Alternatively, cells comprising the tumorvasculature may be targeted. Protein-based recognition molecules candirect the conjugate to specific types of cells such as specifictargeting to hepatocytes in the liver as opposed to Kupffer cells. Inother cases, protein-based recognition molecules can direct theconjugate to cells of the reticular endothelial or lymphatic system, orto professional phagocytic cells such as macrophages or eosinophils. (Insuch cases the conjugate itself might also be an effective deliverysystem, without the need for specific targeting).

In still other embodiments, the protein based recognition molecule cantarget the conjugate to a location within the cell, such as the nucleus,the cytoplasm, or the endosome, for example. In specific embodiments,the protein based recognition molecule can enhance cellular binding toreceptors, or cytoplasmic transport to the nucleus and nuclear entry orrelease from endosomes or other intracellular vesicles.

In specific embodiments the protein based recognition molecules includeantibodies, proteins and peptides or peptide mimics.

In a preferred embodiment, the protein based recognition moleculecomprises a sulfhydryl group and the protein based recognition moleculeis conjugated to the Linker-Drug moiety by forming a covalent bond viathe sulfhydryl group and a functional group of the Linker-Drug moiety.

Exemplary antibodies or antibodies derived from Fab, Fab2, scFv or camelantibody heavy-chain fragments specific to the cell surface markers,include, but are not limited to, 5T4, AOC3, ALK, AXL, C242, C4.4a,CA-125, CCL11, CCR 5, CD2, CD3, CD4, CD5, CD15, CA15-3, CD18, CD19,CA19-9, CDH6, CD20, CD22, CD23, CD25, CD28, CD30, CD31, CD33, CD37,CD38, CD40, CD41, CD44, CD44 v6, CD51, CD52, CD54, CD56, CD62E, CD62P,CD62L, CD70, CD74, CD79-B, CD80, CD125, CD138, CD141, CD147, CD152, CD154, CD326, CEA, CEACAM-5, clumping factor, CTLA-4, CXCR2, EGFR (HER1),ErbB2, ErbB3, EpCAM, EPHA2, EPHB2, EPHB4, FGFR (i.e. FGFR1, FGFR2,FGFR3, FGFR4), FLT3, folate receptor, FAP, GD2, GD3, GPNMB, GCC(GUCY2C), HGF, HER2, HER3, HMI.24, ICAM, ICOS-L, IGF-1 receptor, VEGFR1,EphA2, TRPV1, CFTR, gpNMB, CA9, Cripto, c-KIT, c-MET, ACE, APP,adrenergic receptor-beta2, Claudine 3, LIV1, LY6E, Mesothelin, MUC1,MUC13, NaPi2b, NOTCH1, NOTCH2, NOTCH3, NOTCH4, RON, ROR1, PD-L1, PD-L2,PTK7, B7-H3, B7-B4, IL-2 receptor, IL-4 receptor, IL-13 receptor,TROP-2, frizzled-7, integrins (including α₄, α_(v)β₃, α_(v)β₅, α_(v)β₆,α₁β₄, α₄β₁, α₄β₇, α₅β₁, α₆β₄, α_(IIIb)β₃ intergins), IFN-α, IFN-γ, IgE,IgE, IGF-1 receptor, IL-1, IL-12, IL-23, IL-13, IL-22, IL-4, IL-5, IL-6,interferon receptor, ITGB2 (CD18), LFA-1 (CD11a), L-selectin (CD62L),mucin, myostatin, NCA-90, NGF, PDGFRα, phosphatidylserine, prostaticcarcinoma cell, Pseudomonas aeruginosa, rabies, RANKL, respiratorysyncytial virus, Rhesus factor, SLAMF7, sphingosine-1-phosphate, TAG-72,T-cell receptor, tenascin C, TGF-1, TGF-β2, TGF-β, TNF-α, TRAIL-R1,TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR2, vimentin, and thelike.

In one embodiment the antibodies or antibody derived from Fab, Fab2,scFv or camel antibody heavy-chain fragments specific to the cellsurface markers include CA-125, C242, CD3, CD19, CD22, CD25, CD30, CD31,CD33, CD37, CD40, CD44, CD51, CD54, CD56, CD62E, CD62P, CD62L, CD70,CD138, CD141, CD326, CEA, CTLA-4, EGFR (HER1), ErbB2, ErbB3, FAP, folatereceptor, IGF-1 receptor, GD3, GPNMB, HGF, HER2, VEGF-A, VEGFR2, VEGFR1,EphA2, EpCAM, 5T4, TAG-72, tenascin C, TRPV1, CFTR, gpNMB, CA9, Cripto,ACE, APP, PDGFR α, phosphatidylserine, prostatic carcinoma cells,adrenergic receptor-beta2, Claudine 3, mucin, MUC1, NaPi2b, B7H3, B7H4,C4.4a, CEACAM-5, MUC13, TROP-2, frizzled-7, Mesothelin, IL-2 receptor,IL-4 receptor, IL-13 receptor and integrins (including α_(v) β₃, α_(v)β₅, α_(v) β₆, α₁ β₄, α₄ β₁, α₅ β₁, α₆ β₄ intergins), tenascin C,TRAIL-R2 and vimentin.

Exemplary antibodies include 3F8, abagovomab, abciximab (REOPRO),adalimumab (HUMIRA), adecatumumab, afelimomab, afutuzumab, alacizumab,ALD518, alemtuzumab (CAMPATH), altumomab, amatuximab, anatumomab,anrukinzumab, apolizumab, arcitumomab (CEA-SCAN), aselizumab, atlizumab(tocilizumab, Actemra, RoActemra), atorolimumab, bapineuzumab,basiliximab (Simulect), bavituximab, bectumomab (LYMPHOSCAN), belimumab(BENLYSTA), benralizumab, bertilimumab, besilesomab (SCINITIMUN),bevacizumab (AVASTIN), biciromab (FIBRISCINT), bivatuzumab,blinatumomab, brentuximab, briakinumab, canakinumab (ILARIS),cantuzumab, capromab, catumaxomab (REMOVAB), CC49, cedelizumab,certolizumab, cetuximab (ERBITUX), citatuzumab, cixutumumab,clenoliximab, clivatuzumab, conatumumab, CR6261, dacetuzumab, daclizumab(ZENAPAX), daratumumab, denosumab (PROLIA), detumomab, dorlimomab,dorlixizumab, ecromeximab, eculizumab (SOLIRIS), edobacomab, edrecolomab(PANOREX), efalizumab (RAPTIVA), efungumab (MYCOGRAB), elotuzumab,elsilimomab, enlimomab, epitumomab, epratuzumab, erlizumab, ertumaxomab(REXOMUN), etaracizumab (ABEGRIN), exbivirumab, fanolesomab(NEUTROSPEC), faralimomab, farletuzumab, felvizumab, fezakinumab,figitumumab, fontolizumab (HuZAF), foravirumab, fresolimumab, galiximab,gantenerumab, gavilimomab, gemtuzumab, girentuximab, glembatumumab,golimumab (SIMPONI), gomiliximab, ibalizumab, ibritumomab, igovomab(INDIMACIS-125), imciromab (MYOSCINT), infliximab (REMICADE),intetumumab, inolimomab, inotuzumab, ipilimumab, iratumumab, keliximab,labetuzumab (CEA-CIDE), lebrikizumab, lemalesomab, lerdelimumab,lexatumumab, libivirumab, lintuzumab, lucatumumab, lumiliximab,mapatumumab, maslimomab, matuzumab, mepolizumab (BOSATRIA), metelimumab,milatuzumab, minretumomab, mitumomab, morolimumab, motavizumab (NUMAX),muromonab-CD3 (ORTHOCLONE OKT3), nacolomab, naptumomab, natalizumab(TYSABRI), nebacumab, necitumumab, nerelimomab, nimotuzumab (THERACIM),nofetumomab, ocrelizumab, odulimomab, ofatumumab (ARZERRA), olaratumab,omalizumab (XOLAIR), ontecizumab, oportuzumab, oregovomab (OVAREX),otelixizumab, pagibaximab, palivizumab (SYNAGIS), panitumumab(VECTIBIX), panobacumab, pascolizumab, pemtumomab (THERAGYN), pertuzumab(OMNITARG), pexelizumab, pintumomab, priliximab, pritumumab, PRO 140,rafivirumab, ramucirumab, ranibizumab (LUCENTIS), raxibacumab,regavirumab, reslizumab, rilotumumab, rituximab (RITUXAN), robatumumab,rontalizumab, rovelizumab (LEUKARREST), ruplizumab (ANTOVA), satumomabpendetide, sevirumab, sibrotuzumab, sifalimumab, siltuximab, siplizumab,solanezumab, sonepcizumab, sontuzumab, stamulumab, sulesomab(LEUKOSCAN), tacatuzumab (AFP-CIDE), tetraxetan, tadocizumab, talizumab,tanezumab, taplitumomab paptox, tefibazumab (AUREXIS), telimomab,tenatumomab, teneliximab, teplizumab, TGN1412, ticilimumab(tremelimumab), tigatuzumab, TNX-650, tocilizumab (atlizumab, ACTEMRA),toralizumab, tositumomab (BEXXAR), trastuzumab (HERCEPTIN),tremelimumab, tucotuzumab, tuvirumab, urtoxazumab, ustekinumab(STELERA), vapaliximab, vedolizumab, veltuzumab, vepalimomab,visilizumab (NUVION), volociximab (HUMASPECT), votumumab, zalutumumab(HuMEX-EGFr), zanolimumab (HuMAX-CD4), ziralimumab and zolimomab.

In some embodiments, the antibodies are directed to cell surface markersfor 5T4, CA-125, CEA, CDH6, CD3, CD19, CD20, CD22, CD30, CD33, CD40,CD44, CD51, CTLA-4, CEACAM5, EpCAM, HER2, EGFR (HER1), FAP, folatereceptor, GCC (GUCY2C), HGF, integrin α_(v)β₃, integrin α₅β₁, IGF-1receptor, GD3, GPNMB, mucin, LIV1, LY6E, mesothelin, MUC1, MUC13, PTK7,phosphatidylserine, prostatic carcinoma cells, PDGFR α, TAG-72, tenascinC, TRAIL-R2, VEGF-A and VEGFR2. In this embodiment the antibodies areabagovomab, adecatumumab, alacizumab, altumomab, anatumomab,arcitumomab, bavituximab, bevacizumab (AVASTIN), bivatuzumab,blinatumomab, brentuximab, cantuzumab, catumaxomab, capromab, cetuximab,citatuzumab, clivatuzumab, conatumumab, dacetuzumab, edrecolomab,epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab,gemtuzumab, glembatumumab, ibritumomab, igovomab, intetumumab,inotuzumab, labetuzumab, lexatumumab, lintuzumab, lucatumumab,matuzumab, mitumomab, naptumomab estafenatox, necitumumab, oportuzumab,oregovomab, panitumumab, pemtumomab, pertuzumab, pritumumab, rituximab(RITUXAN), rilotumumab, robatumumab, satumomab, sibrotuzumab,taplitumomab, tenatumomab, tenatumomab, ticilimumab (tremelimumab),tigatuzumab, trastuzumab (HERCEPTIN), tositumomab, tremelimumab,tucotuzumab celmoleukin, volociximab and zalutumumab.

In specific embodiments the antibodies directed to cell surface markersfor HER2 are pertuzumab or trastuzumab and for EGFR (HER1) the antibodyis cetuximab or panitumumab; and for CD20 the antibody is rituximab andfor VEGF-A is bevacizumab and for CD-22 the antibody is epratuzumab orveltuzumab and for CEA the antibody is labetuzumab.

Exemplary peptides or peptide mimics include integrin targeting peptides(RGD peptides), LHRH receptor targeting peptides, ErbB2 (HER2) receptortargeting peptides, prostate specific membrane bound antigen (PSMA)targeting peptides, lipoprotein receptor LRP1 targeting, ApoE proteinderived peptides, ApoA protein peptides, somatostatin receptor targetingpeptides, chlorotoxin derived peptides, and bombesin.

In specific embodiments the peptides or peptide mimics are LHRH receptortargeting peptides and ErbB2 (HER2) receptor targeting peptides

Exemplary proteins comprise insulin, transferrin, fibrinogen-gammafragment, thrombospondin, claudin, apolipoprotein E, Affibody moleculessuch as, for example, ABY-025, Ankyrin repeat proteins, ankyrin-likerepeats proteins and synthetic peptides.

In some embodiments, the protein-drug conjugates comprise broad spectrumcytotoxins in combination with cell surface markers for HER2 such aspertuzumab or trastuzumab; for EGFR such as cetuximab and panitumumab;for CEA such as labetuzumab; for CD20 such as rituximab; for VEGF-A suchas bevacizumab; or for CD-22 such as epratuzumab or veltuzumab.

In other embodiments, the protein-drug conjugates or protein conjugatesused in the disclosure comprise combinations of two or more proteinbased recognition molecules, such as, for example, combination ofbispecific antibodies directed to the EGF receptor (EGFR) on tumor cellsand to CD3 and CD28 on T cells; combination of antibodies or antibodyderived from Fab, Fab2, scFv or camel antibody heavy-chain fragments andpeptides or peptide mimetics; combination of antibodies or antibodyderived from Fab, Fab2, scFv or camel antibody heavy-chain fragments andproteins; combination of two bispecific antibodies such as CD3×CD19 plusCD28×CD22 bispecific antibodies.

In other embodiments, the protein-drug conjugates or protein conjugatesused in the disclosure comprise protein based recognition molecules areantibodies against antigens, such as, for example, Trastuzumab,Cetuximab, Rituximab, Bevacizumab, Epratuzumab, Veltuzumab, Labetuzumab,B7-H4, B7-H3, CA125, CDH6, CD33, CXCR2, CEACAM5, EGFR, FGFR1, FGFR2,FGFR3, FGFR4, GCC (GUCY2C), HER2, LIV1, LY6E, NaPi2b, c-Met, mesothelin,NOTCH1, NOTCH2, NOTCH3, NOTCH4, PD-L1, PTK7, c-Kit, MUC1, MUC13. and5T4.

In a specific embodiment, the protein-drug conjugates or proteinconjugates of the disclosure comprise protein based recognitionmolecules which are antibodies against 5T4, such as, for example ahumanized anti-5T4 scFvFc antibody.

Examples of suitable 5T4 targeting ligands or immunoglobulins includethose which are commercially available, or have been described in thepatent or non-patent literature, e.g., U.S. Pat. Nos. 8,044,178,8,309,094, 7,514,546, EP1036091 (commercially available as TroVax™,Oxford Biomedica), EP2368914A1, WO 2013041687 A1 (Amgen), US2010/0173382, and P. Sapra, et al., Mol. Cancer Ther. 2013, 12:38-47. Ananti-5T4 antibody is disclosed in U.S. Provisional Application No.61/877,439, filed Sep. 13, 2013 and U.S. Provisional Application No.61/835,858, filed Jun. 17, 2013. The contents of each of the patentdocuments and scientific publications are herein incorporated byreference in their entireties.

As used herein, the term “5T4 antigen-binding portion” refers to apolypeptide sequence capable of selectively binding to a 5T4 antigen. Inexemplary conjugates, the 5T4 antigen-binding portion generallycomprises a single chain scFv-Fc form engineered from an anti-5T4antibody. A single-chain variable fragment (scFv-Fc) is a fusion proteinof the variable regions of the heavy (VH) and light chains (VL) of animmunoglobulin, connected with a linker peptide, and further connectedto an Fc region comprising a hinge region and CH2 and CH3 regions of anantibody (any such combinations of antibody portions with each other orwith other peptide sequences is sometimes referred to herein as an“immunofusion” molecule). Within such a scFvFc molecule, the scFvsection may be C-terminally linked to the N-terminus of the Fc sectionby a linker peptide.

In other specific embodiments, the protein-drug conjugates or proteinconjugates of the disclosure comprise protein based recognitionmolecules which are Her-2 or NaPi2b antibodies.

For example the Her-2 antibody suitable for the conjugate or scaffold ofthe disclosure comprises a variable heavy chain complementaritydetermining region 1 (CDRH1) comprising the amino acid sequenceFTFSSYSMN (SEQ ID NO: 25); a variable heavy chain complementaritydetermining region 2 (CDRH2) comprising the amino acid sequenceYISSSSSTIYYADSVKG (SEQ ID NO: 26); a variable heavy chaincomplementarity determining region 3 (CDRH3) comprising the amino acidsequence GGHGYFDL (SEQ ID NO: 27); a variable light chaincomplementarity determining region 1 (CDRL1) comprising the amino acidsequence RASQSVSSSYLA (SEQ ID NO: 28); a variable light chaincomplementarity determining region 2 (CDRL2) comprising the amino acidsequence GASSRAT (SEQ ID NO: 21); and a variable light chaincomplementarity determining region 3 (CDRL3) comprising the amino acidsequence QQYHHSPLT (SEQ ID NO: 29) (see, e.g., US20150366987(A1)published Dec. 24, 2015).

For example, the NaPi2b antibody suitable for the conjugate or scaffoldof the disclosure comprises a variable light chain complementaritydetermining region 1 (CDRL1) comprising the amino acid sequenceSASQDIGNFLN (SEQ ID NO: 8); a variable light chain complementaritydetermining region 2 (CDRL2) comprising the amino acid sequence YTSSLYS(SEQ ID NO: 9); a variable light chain complementarity determiningregion 3 (CDRL3) comprising the amino acid sequence QQYSKLPLT (SEQ IDNO: 10); a variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence GYTFTGYNIH (SEQ ID NO: 5); avariable heavy chain complementarity determining region 2 (CDRH2)comprising the amino acid sequence AIYPGNGDTSYKQKFRG (SEQ ID NO: 6); anda variable heavy chain complementarity determining region 3 (CDRH3)comprising the amino acid sequence GETARATFAY (SEQ ID NO: 7) (see, e.g.,co-pending application U.S. Ser. No. 15/457,574 filed Mar. 13, 2017).

PBRM-Drug Conjugates

Conjugates of the disclosure comprise one or more occurrences of D,where D is a therapeutic agent, e.g., a drug, wherein the one or moreoccurrences of D may be the same or different.

In certain other embodiments, one or more occurrences of PBRM isattached to the Linker-Drug moiety, wherein the one or more occurrencesof PBRM may be the same or different. In certain other embodiments, oneor more Linker-Drug moieties that contains one or more occurrences of Dare connected to one PBRM (e.g., an antibody).

In one embodiment, D is a) an auristatin compound; (b) a calicheamicincompound; (c) a duocarmycin compound; (d) a topoisomerase inhibitor, (e)a pyrrolobenzodiazepine compound; (f) a vinca compound; (g) a proteinsynthesis inhibitor; (h) a RNA polymerase inhibitor; (i) a tubulinbinding compound; (j) a NAMPT inhibitor or an analog thereof.

In certain embodiment, D is (a) an auristatin compound; (b) acalicheamicin compound; (c) a duocarmycin compound; (d) a camptothecincompound, (e) a pyrrolobenzodiazepine compound; (f) a vinca compound; oran analog thereof.

For example, the auristatin compound is auristatin, dolastatin,monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), auristatinF, AF HPA, MMAF HPA, or phenylenediamine (AFP).

For example, the duocarmycin or an analog thereof is duocarmycin A,duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2,duocarmycin D, duocarmycin SA, CC-1065, adozelesin, bizelesin, orcarzelesin.

For example, the camptothecin compound is camptothecin, CPT-11(irinotecan), SN-38, or topotecan.

For example, the pyrrolobenzodiazepine compound is apyrrolobenzodiazepine monomer, a symmetrical pyrrolobenzodiazepine dimeror an unsymmetrical pyrrolobenzodiazepine dimer.

The PBRM-drug conjugate of the disclosure comprise a PBRM that has amolecular weight of about 40 kDa or greater (e.g., 60 kDa or greater; 80kDa or greater; 100 kDa or greater; 120 kDa or greater; 140 kDa orgreater; 160 kDa or greater; 180 kDa or greater; or 200 kDa or greater,or about 40-200 kDa, 40-180 kDa, 40-140 kDa, 60-200 kDa, 60-180 kDa,60-140 kDa, 80-200 kDa, 80-180 kDa, 80-140 kDa, 100-200 kDa, 100-180kDa, or 100-140 kDa).

For example, the PBRM has a molecular weight of about 40 kDa or greater(e.g., 60 kDa or greater; 80 kDa or greater; 100 kDa or greater; 120 kDaor greater; 140 kDa or greater; 160 kDa or greater; 180 kDa or greater;or 200 kDa or greater, or about 40-200 kDa, 40-180 kDa, 40-140 kDa,60-200 kDa, 60-180 kDa, 60-140 kDa, 80-200 kDa, 80-180 kDa, 80-140 kDa,100-200 kDa, 100-180 kDa, or 100-140 kDa) and has a sulfhydryl (i.e.,—SH or thiol) group.

For example, the total number of sulfide bonds formed between the PHFand the PBRM (or total number of attachment points) is 10 or less.

For example, for conjugation with one or more Linker-Drug moieties, thePBRM has a molecular weight of 40 kDa or greater (e.g., 60 kDa orgreater, 80 kDa or greater, 100 kDa or greater, 120 kDa or greater, 140kDa or greater, 160 kDa or greater or 180 kDa or greater, or about40-200 kDa, 40-180 kDa, 40-140 kDa, 60-200 kDa, 60-180 kDa, 60-140 kDa,80-200 kDa, 80-180 kDa, 80-140 kDa, 100-200 kDa, 100-180 kDa, or 100-140kDa

For example, for conjugation with one or more Linker-Drug moieties, thePBRM has a molecular weight of 40 kDa to 200 kDa.

For example, for conjugation with one or more Linker-Drug moieties, thePBRM has a molecular weight of 40 kDa to 80 kDa.

PBRMs in this molecular weight range include, but are not limited to,for example, antibody fragments, such as, for example, Fabs.

For example, for conjugation with one or more Linker-Drug moieties, thePBRM has a molecular weight of 60 kDa to 120 kDa.

PBRMs in this molecular weight range include, but are not limited to,for example, camelids, Fab2, scFvFc, and the like.

For example, for conjugation with one or more Linker-Drug moieties, thePBRM has a molecular weight of 140 kDa to 180 kDa.

PBRMs in this molecular weight range include, but are not limited to,for example, full length antibodies, such as, IgG, IgM.

These targeting ligands, the linkers and the drug or prodrug fragmentsdescribed herein can be assembled into the conjugate or scaffold of thedisclosure, for example according to the disclosed techniques andmethods. Therapeutic and targeting conjugates of the disclosure, andmethods for producing them, are described below by way of non-limitingexample.

For example, the total number of sulfide bonds formed between theLinker-Drug moiety and the PBRM (or total number of attachment points)is 14 or less.

For example, the ratio between the Linker-Drug moiety and the PBRM isgreater than 1:1 and less than or equal to 14:1.

For example, the ratio between Linker-Drug moiety is about 14:1, 12:1,13:1, 12:1, 11:1, 10;1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

For example, the ratio between Linker-Drug moiety and the PBRM isbetween 2:1 and 10:1.

For example, the ratio between Linker-Drug moiety and the PBRM is about10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

For example, the ratio between Linker-Drug moiety and the PBRM isbetween 2:1 and 4:1.

For example, the ratio between Linker-Drug moiety and the PBRM is about4:1, 3:1, or 2:1.

In some embodiments, the Linker-Drug moiety is conjugated with PBRMs byutilizing cysteine-based bioconjugation strategy. See, e.g.,WO2010100430 and U.S. Pat. No. 7,595,292, the contents of which arehereby incorporated by reference in their entireties. In one embodiment,one or more Linker-Drug moieties conjugate with a PBRM (e.g., anantibody) via cysteines in the antibody hinge region. Without wishing tobe bound by the theory, the resulting conjugate is stabilized throughthe formation of inter-chain bridge structures.

Accordingly, the disclosure also relates to a Linker-Drug moietycomprising at least two moieties, in which each moiety is capable ofconjugation to a thiol group from an amino acid (e.g., cysteine) in aPBRM so as to form a protein-Linker-Drug conjugate.

In embodiments, one or more free thiol groups of a PBRM are produced byreducing a protein. The one or more free thiol groups of the PBRM thenreact with one or more Linker-Drug moieties that are capable ofconjugation to a thiol group from an amino acid so as to conjugate thePBRM with the Linker-Drug moiety. In one embodiment, the at least twomoieties connected to the PBRM are maleimide groups.

In embodiments, the free thiol groups of the PBRM that are used for theconjugation are derived from a disulfide bridge of a native protein or adisulfide bridge of a protein complex consisting of two or more proteinchains connected by the disulfide bridge. A disulfide bridge may beintrachain or interchain bridge. Alternatively, the free thiol groups ofthe PBRM are from cysteines or the unpaired thiol groups of the nativeprotein that are not involved in inter or intra disulfide bridgeformation.

Disulfide bonds can be reduced, for example, with dithiothreitol,mercaptoethanol, tris-carboxyethylphosphine, dehydroascorbic acid,copper sulfate, using conventional methods. A protein can contain one ormore disulfide bridges. Reduction to give free thiol groups can becontrolled to reduce one or more specific disulfide bridges in aprotein. Depending on the extent of disulfide reduction and thestoichiometry of the moieties on the Linker-Drug moiety, it is possibleto conjugate one or more Linker-Drug moieties to the protein.Immobilized reducing agents may be used if it is desired to reduce lessthan the total number of disulfides, as can partial reduction usingdifferent reaction conditions or the addition of denaturants.

For example, for conjugating of the Linker-Drug moiety, a PBRM has amolecular weight of 40 kDa or greater (e.g., 60 kDa or greater; 80 kDaor greater; or 100 kDa or greater; 120 kDa or greater; 140 kDa orgreater; 160 kDa or greater or 180 kDa or greater). In this embodimentthe ratio of PBRM per Linker-Drug moiety is between about 1:1 and about1:10, between about 1:1 and about 1:9, between about 1:1 and about 1:8,between about 1:1 and about 1:7, between about 1:1 and about 1:6,between about 1:1 and about 1:5, between about 1:1 and about 1:4,between about 1:1 and about 1:3, between about 1:1 and about 1:2,between about 1:2 and about 1:6, between about 1:2 and about 1:5,between about 1:2 and about 1:4 or between about 1:2 and about 1:3.

PBRMs in this molecular weight range include, but are not limited to,for example, full length antibodies, such as, IgG, IgM.

For example, for conjugation with one or more Linker-Drug moieties aPBRM has a molecular weight of 60 kDa to 120 kDa. In this embodiment theratio of PBRM per Linker-Drug moiety is between about 1:1 and about1:10, between about 1:1 and about 1:9, between about 1:1 and about 1:8,between about 1:1 and about 1:7, between about 1:1 and about 1:6,between about 1:1 and about 1:5, between about 1:1 and about 1:4,between about 1:1 and about 1:3, between about 1:1 and about 1:2,between about 1:2 and about 1:6, between about 1:2 and about 1:5,between about 1:2 and about 1:4 or between about 1:2 and about 1:3.

PBRMs in this molecular weight range include, but are not limited to,for example, antibody fragments such as, for example Fab2, scFcFv andcamelids.

For example, for conjugation with one or more Linker-Drug moieties aPBRM has a molecular weight of 40 kDa to 80 kDa. In this embodiment theratio of PBRM per Linker-Drug moiety is between about 1:1 and about1:10, between about 1:1 and about 1:9, between about 1:1 and about 1:8,between about 1:1 and about 1:7, between about 1:1 and about 1:6,between about 1:1 and about 1:5, between about 1:1 and about 1:4,between about 1:1 and about 1:3, between about 1:1 and about 1:2,between about 1:2 and about 1:6, between about 1:2 and about 1:5,between about 1:2 and about 1:4 or between about 1:2 and about 1:3.

PBRMs in this molecular weight range include, but are not limited to,for example, antibody fragments, such as, Fabs.

In another aspect, the disclosure features a saffold useful to conjugatewith either or both of a protein based recognition-molecule (PBRM) and atherapeutic agent (D), e.g., the scaffold of any of Formulae (II)-(IX)disclosed herein.

In some embodiments, the drug-carrying scaffolds (i.e., without linkingto a PBRM), described herein each typically have a polydispersity index(PDI) of 1.

Conjugates and scaffolds disclosed herein can be purified (i.e., removalof any starting materials) by extensive diafiltration. If necessary,additional purification by size exclusion chromatography can beconducted to remove any aggregated conjugates. In general, theconjugates as purified typically contain less than 5% (e.g., <2% w/w)aggregated conjugates as determined by SEC; less than 0.5% (e.g., <0.1%w/w) free (unconjugated) drug as determined by RP-HPLC; less than 1%drug carrying-peptide-containing scaffolds as determined by SEC and lessthan 2% (e.g., <1% w/w) unconjugated PBRM as determined by HIC-HPLC.

Tables B and C below provide examples of the drugcarrying-peptide-containing scaffolds and the conjugates of thedisclosure respectively.

TABLE B Scaffold No. Structure Scaffold No. 7 Example 1

Scaffold No. 17 Example 2

Scaffold No. 24 Example 6

Scaffold No. 27 Example 8

Scaffold No. 30 Example 10

Scaffold No. 33 Example 12

Scaffold No 42 Example 17

Scaffold No 45 Example 19

Scaffold No. 47 Example 21

Scaffold No. 49 Example 22

Scaffold No. 51 Example 23

Scaffold No. 54 Example 25

Scaffold No. 56 Example 26

Scaffold No. 58 Example 27

Scaffold No. 60 Example 28

Scaffold No. 65 Example 29

Scaffold No. 75 Example 32 Example 36

Scaffold No. 77 Example 33

Scaffold No. 84 Example 38

TABLE C Conjugate No. Structure Conjugate No. 25 Example 7

Conjugate No. 28 Example 9

Conjugate No. 31 Example 11

Conjugate No. 34 Example 13

Conjugate No. 43 Example 18

Conjugate No. 46 Example 20

Conjugate No. 48 Example 21

Conjugate No. 50 Example 22

Conjugate No. 52 Example 23

Conjugate No. 53 Example 24

Conjugate No. 55 Example 25

Conjugate No. 57 Example 26

Conjugate No. 59 Example 27

Conjugate No. 61 Example 28

Conjugate No. 66 Example 29 Conjugate 79, Example 35

Conjugate No. 67 Example 30

Conjugate No. 68 Example 31

Conjugate No. 76 Example 33 Conjugate No. 83 Example 37

Conjugate No. 78 Example 34 Conjugate No. 85 Example 38

In some embodiments, the protein-drug conjugates are conjugates ofFormula (XXX):

wherein each R_(A) is

In other embodiments, the protein-drug conjugates are conjugates ofFormula (XXX):

wherein each R_(A) is

For example, the protein-drug conjugate is of Formula (XXX), whereineach R_(A) is

For example, the protein-drug conjugate is of Formula (XXX), whereineach R_(A) is

For example, the protein-drug conjugate is of Formula (XXX), whereineach R_(A) is

For example, the protein-drug conjugate is of Formula (XXX), whereineach R_(A) is

For example, the protein-drug conjugate is of Formula (XXX), whereineach R_(A) is

For example, the protein-drug conjugate is of Formula (XXX), whereineach R_(A) is

For example, the protein-drug conjugate is of Formula (XXX), whereineach R_(A) is

For example, the protein-drug conjugate is of Formula (XXX), whereineach R_(A) is:

For example, the protein-drug conjugate is of Formula (XXX), whereineach R_(A) is

In some embodiments, the protein-drug conjugates are conjugates ofFormula (XXXII-1), (XXXII-2), (XXXII-3) or (XXXII-4):

In some embodiments, the protein-drug conjugates are conjugates ofFormula (XXXIII):

wherein each R_(B) is:

In some embodiments, in the protein-drug conjugates are conjugates ofFormula (XXXII-1), (XXXII-2), (XXXII-3), (XXXII-4) or (XXXIII), thevariable -L^(D)-D is:

In other embodiments, the protein-drug conjugates are conjugates ofFormula (XXXIV-1) (XXXIV-2), (XXXXIV-3) or (XXXIV-4):

Pharmaceutical Compositions

Also included are pharmaceutical compositions comprising one or moreconjugates as disclosed herein in an acceptable carrier, such as astabilizer, buffer, and the like. The conjugates can be administered andintroduced into a subject by standard means, with or withoutstabilizers, buffers, and the like, to form a pharmaceuticalcomposition. Administration may be topical (including ophthalmic and tomucous membranes including vaginal and rectal delivery), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral administration including intravenous, intraarterial,subcutaneous, intraperitoneal or intramuscular injection or infusion orintracranial, e.g., intrathecal or intraventricular, administration. Theconjugates can be formulated and used as sterile solutions and/orsuspensions for injectable administration; lyophilized powders forreconstitution prior to injection/infusion; topical compositions; astablets, capsules, or elixirs for oral administration; or suppositoriesfor rectal administration, and the other compositions known in the art.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemicadministration, into a cell or subject, including for example a human.Suitable forms, in part, depend upon the use or the route of entry, forexample oral, inhaled, transdermal, or by injection/infusion. Such formsshould not prevent the composition or formulation from reaching a targetcell (i.e., a cell to which the drug is desirable for delivery). Forexample, pharmacological compositions injected into the blood streamshould be soluble. Other factors are known in the art, and includeconsiderations such as toxicity and forms that prevent the compositionor formulation from exerting its effect.

By “systemic administration” is meant in vivo systemic absorption oraccumulation of the conjugate in the blood stream followed bydistribution throughout the entire body. Administration routes that leadto systemic absorption include, without limitation: intravenous,subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary, andintramuscular. Each of these administration routes exposes theconjugates to an accessible diseased tissue. The rate of entry of anactive agent into the circulation has been shown to be a function ofmolecular weight or size. The use of a conjugate of this disclosure canlocalize the drug delivery in certain cells, such as cancer cells viathe specificity of PBRMs.

A “pharmaceutically acceptable formulation” means a composition orformulation that allows for the effective distribution of the conjugatesin the physical location most suitable for their desired activity. Inone embodiment, effective delivery occurs before clearance by thereticuloendothelial system or the production of off-target binding whichcan result in reduced efficacy or toxicity. Non-limiting examples ofagents suitable for formulation with the conjugates include:P-glycoprotein inhibitors (such as Pluronic P85), which can enhanceentry of active agents into the CNS; biodegradable polymers, such aspoly (DL-lactide-coglycolide) microspheres for sustained releasedelivery after intracerebral implantation; and loaded nanoparticles,such as those made of polybutylcyanoacrylate, which can deliver activeagents across the blood brain barrier and can alter neuronal uptakemechanisms.

Also included herein are pharmaceutical compositions prepared forstorage or administration, which include a pharmaceutically effectiveamount of the desired conjugates in a pharmaceutically acceptablecarrier or diluent. Acceptable carriers, diluents, and/or excipients fortherapeutic use are well known in the pharmaceutical art. For example,buffers, preservatives, bulking agents, dispersants, stabilizers, dyes,can be provided. In addition, antioxidants and suspending agents can beused Examples of suitable carriers, diluents and/or excipients include,but are not limited to: (1) Dulbecco's phosphate buffered saline, pHabout 6.5, which would contain about 1 mg/ml to 25 mg/ml human serumalbumin, (2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose.

The term “pharmaceutically effective amount”, as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic or combination of therapeuticsselected for administration. Pharmaceutically effective amounts for agiven situation can be determined by routine experimentation that iswithin the skill and judgment of the clinician. In a preferred aspect,the disease or condition to can be treated via gene silencing.

For any conjugate, the pharmaceutically effective amount can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually rats, mice, rabbits, dogs, or pigs.The animal model may also be used to determine the appropriateconcentration range and route of administration. Such information canthen be used to determine useful doses and routes for administration inhumans. Therapeutic/prophylactic efficacy and toxicity may be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

For example, a drug or its derivatives, drug-conjugates or PBRM-drugconjugates can be evaluated for their ability to inhibit tumor growth inseveral cell lines using Cell titer Glo. Dose response curves can begenerated using SoftMax Pro software and IC₅₀ values can be determinedfrom four-parameter curve fitting. Cell lines employed can include thosewhich are the targets of the PBRM and a control cell line that is notthe target of the PBRM contained in the test conjugates.

In one embodiment, the conjugates are formulated for parenteraladministration by injection including using conventional catheterizationtechniques or infusion. Formulations for injection may be presented inunit dosage form, e.g., in ampules or in multi-dose containers, with anadded preservative. The conjugates can be administered parenterally in asterile medium. The conjugate, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives, andbuffering agents can be dissolved in the vehicle. The term “parenteral”as used herein includes percutaneous, subcutaneous, intravascular (e.g.,intravenous), intramuscular, or intrathecal injection or infusiontechniques and the like. In addition, there is provided a pharmaceuticalformulation comprising conjugates and a pharmaceutically acceptablecarrier. One or more of the conjugates can be present in associationwith one or more non-toxic pharmaceutically acceptable carriers and/ordiluents and/or adjuvants, and if desired other active ingredients.

The sterile injectable preparation can also be a sterile injectablesolution or suspension in a non-toxic parentally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, a bland fixed oil can be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The conjugates and compositions described herein may be administered inappropriate form, preferably parenterally, more preferablyintravenously. For parenteral administration, the conjugates orcompositions can be aqueous or nonaqueous sterile solutions, suspensionsor emulsions. Propylene glycol, vegetable oils and injectable organicesters, such as ethyl oleate, can be used as the solvent or vehicle. Thecompositions can also contain adjuvants, emulsifiers or dispersants.

Dosage levels of the order of from between about 0.001 mg and about 140mg per kilogram of body weight per day are useful in the treatment ofthe above-indicated conditions (between about 0.05 mg and about 7 g persubject per day). In some embodiments, the dosage administered to apatient is between about 0.001 mg/kg to about 100 mg/kg of the subject'sbody weight. In some embodiments, the dosage administered to a patientis between about 0.01 mg/kg to about 15 mg/kg of the subject's bodyweight. In some embodiments, the dosage administered to a patient isbetween about 0.1 mg/kg and about 15 mg/kg of the subject's body weight.In some embodiments, the dosage administered to a patient is betweenabout 0.1 mg/kg and about 20 mg/kg of the subject's body weight. In someembodiments, the dosage administered is between about 0.1 mg/kg to about5 mg/kg or about 0.1 mg/kg to about 10 mg/kg of the subject's bodyweight. In some embodiments, the dosage administered is between about 1mg/kg to about 15 mg/kg of the subject's body weight. In someembodiments, the dosage administered is between about 1 mg/kg to about10 mg/kg of the subject's body weight. The amount of conjugate that canbe combined with the carrier materials to produce a single dosage formvaries depending upon the host treated and the particular mode ofadministration. Dosage unit forms can generally contain from betweenabout 0.001 mg and about 100 mg; between about 0.01 mg and about 75 mg;or between about 0.01 mg and about 50 mg; or between about 0.01 mg andabout 25 mg; of a conjugate.

For intravenous administration, the dosage levels can comprise rangesdescribed in the preceding paragraphs, or from about 0.01 to about 200mg of a conjugate per kg of the animal's body weight. In one aspect, thecomposition can include from about 1 to about 100 mg of a conjugate perkg of the animal's body weight. In another aspect, the amountadministered will be in the range from about 0.1 to about 25 mg/kg ofbody weight of a compound.

In some embodiments, the conjugates can be administered are as follows.The conjugates can be given daily for about 5 days either as an i.v.,bolus each day for about 5 days, or as a continuous infusion for about 5days.

Alternatively, the conjugates can be administered once a week for sixweeks or longer. As another alternative, the conjugates can beadministered once every two or three weeks. Bolus doses are given inabout 50 to about 400 ml of normal saline to which about 5 to about 10ml of human serum albumin can be added. Continuous infusions are givenin about 250 to about 500 ml of normal saline, to which about 25 toabout 50 ml of human serum albumin can be added, per 24 hour period.

In some embodiments, about one to about four weeks after treatment, thepatient can receive a second course of treatment. Specific clinicalprotocols with regard to route of administration, excipients, diluents,dosages, and times can be determined by the skilled artisan as theclinical situation warrants.

In other embodiments, the therapeutically effective amount may beprovided on another regular schedule, i.e., daily, weekly, monthly, oryearly basis or on an irregular schedule with varying administrationdays, weeks, months, etc. Alternatively, the therapeutically effectiveamount to be administered may vary. In one embodiment, thetherapeutically effective amount for the first dose is higher than thetherapeutically effective amount for one or more of the subsequentdoses. In another embodiment, the therapeutically effective amount forthe first dose is lower than the therapeutically effective amount forone or more of the subsequent doses. Equivalent dosages may beadministered over various time periods including, but not limited to,about every 2 hours, about every 6 hours, about every 8 hours, aboutevery 12 hours, about every 24 hours, about every 36 hours, about every48 hours, about every 72 hours, about every week, about every two weeks,about every three weeks, about every month, and about every two months.The number and frequency of dosages corresponding to a completed courseof therapy will be determined according to the recommendations of therelevant regulatory bodies and judgment of a health-care practitioner.The therapeutically effective amounts described herein refer to totalamounts administered for a given time period; that is, if more than onedifferent conjugate described herein is administered, thetherapeutically effective amounts correspond to the total amountadministered. It is understood that the specific dose level for aparticular subject depends upon a variety of factors including theactivity of the specific conjugate, the age, body weight, generalhealth, sex, diet, time of administration, route of administration, andrate of excretion, combination with other active agents, and theseverity of the particular disease undergoing therapy.

In some embodiments, a therapeutically effective amount of a conjugatedisclosed herein relates generally to the amount needed to achieve atherapeutic objective. As noted above, this may be a binding interactionbetween the antibody and its target antigen that, in certain cases,interferes with the functioning of the target. The amount required to beadministered will furthermore depend on the binding affinity of theantibody for its specific antigen, and will also depend on the rate atwhich an administered antibody is depleted from the free volume othersubject to which it is administered. Common ranges for therapeuticallyeffective dosing of conjugates disclosed herein may be, by way ofnonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kgbody weight, from about 0.1 mg/kg body weight to about 100 mg/kg bodyweight or from about 0.1 mg/kg body weight to about 150 mg/kg bodyweight. Common dosing frequencies may range, for example, from twicedaily to once a month (e.g., once daily, once weekly; once every otherweek; once every 3 weeks or monthly). For example, conjugates disclosedherein can be administered (e.g., as a single dose weekly, every 2weeks, every 3 weeks, or monthly) at about 0.1 mg/kg to about 20 mg/kg(e.g., 0.2 mg/kg, 0.5 mg/kg, 0.67 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg,12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19mg/kg, or 20 mg/kg). For example, conjugates disclosed herein can beadministered (e.g., as a single dose weekly, every 2 weeks, every 3weeks, or monthly) at about 0.1 mg/kg to about 20 mg/kg (e.g., 0.2mg/kg, 0.5 mg/kg, 0.67 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg,13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 19mg/kg, or 20 mg/kg) for treating cancer.

For administration to non-human animals, the conjugates can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water so that the animal takes ina therapeutically appropriate quantity of the conjugates along with itsdiet. It can also be convenient to present the conjugates as a premixfor addition to the feed or drinking water.

The conjugates can also be administered to a subject in combination withother therapeutic compounds to increase the overall therapeutic effect.The use of multiple compounds to treat an indication can increase thebeneficial effects while reducing the presence of side effects. In someembodiments, the conjugates are used in combination withchemotherapeutic agents, such as those disclosed in U.S. Pat. No.7,303,749. In other embodiments the chemotherapeutic agents, include,but are not limited to letrozole, oxaliplatin, docetaxel, 5-FU,lapatinib, capecitabine, leucovorin, erlotinib, pertuzumab, bevacizumab,and gemcitabine. The present disclosure also provides pharmaceuticalkits comprising one or more containers filled with one or more of theconjugates and/or compositions of the present disclosure, including, oneor more chemotherapeutic agents. Such kits can also include, forexample, other compounds and/or compositions, a device(s) foradministering the compounds and/or compositions, and writteninstructions in a form prescribed by a governmental agency regulatingthe manufacture, use or sale of pharmaceuticals or biological products.The compositions described herein can be packaged as a single dose orfor continuous or periodic discontinuous administration. For continuousadministration, a package or kit can include the conjugates in eachdosage unit (e.g., solution or other unit described above or utilized indrug delivery), and optionally instructions for administering the dosesdaily, weekly, or monthly, for a predetermined length of time or asprescribed. If varying concentrations of a composition, of thecomponents of the composition, or the relative ratios of the conjugatesor agents within a composition over time is desired, a package or kitmay contain a sequence of dosage units which provide the desiredvariability.

A number of packages or kits are known in the art for dispensingpharmaceutical agents for periodic oral use. In one embodiment, thepackage has indicators for each period. In another embodiment, thepackage is a labeled blister package, dial dispenser package, or bottle.The packaging means of a kit may itself be geared for administration,such as a syringe, pipette, eye dropper, or other such apparatus, fromwhich the formulation may be applied to an affected area of the body,injected into a subject, or even applied to and mixed with the othercomponents of the kit.

Methods of Use

Methods of Treating

In certain preferred embodiments, the protein-drug conjugate of thedisclosure are used in methods of treating animals (preferably mammals,most preferably humans and includes males, females, infants, childrenand adults). In one embodiment, the conjugates of the present disclosuremay be used in a method of treating animals which comprisesadministering to the animal a biodegradable biocompatible conjugate ofthe disclosure. For example, conjugates of the disclosure can beadministered in the form of soluble linear polymers, copolymers,conjugates, colloids, particles, gels, solid items, fibers, films, etc.Biodegradable biocompatible conjugates disclosed herein can be used asdrug carriers and drug carrier components, in systems of controlled drugrelease, preparations for low-invasive surgical procedures, etc.Pharmaceutical formulations can be injectable, implantable, etc.

In yet another aspect, the disclosure provides a method of treating adisease or disorder in a subject in need thereof, comprisingadministering to the subject an efficient amount of at least oneconjugate of the disclosure; wherein said conjugate releases one or moretherapeutic agents upon biodegradation.

In another embodiment the conjugates can be administered in vitro, invivo and/or ex vivo to treat patients and/or to modulate the growth ofselected cell populations including, for example, cancer. In someembodiments, the particular types of cancers that can be treated withthe conjugates include, but are not limited to: (1) solid tumors,including but not limited to fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer,pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostatecancer, esophogeal cancer, stomach cancer, oral cancer, nasal cancer,throat cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicularcancer, small cell lung carcinoma, non-small cell lung carcinoma,bladder carcinoma, lung cancer, epithelial carcinoma, glioma,glioblastoma, multiforme astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, skin cancer, melanoma,neuroblastoma, and retinoblastoma; (2) blood-borne cancers, includingbut not limited to acute lymphoblastic leukemia “ALL”, acutelymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,acute myeloblastic leukemia “AML”, acute promyelocytic leukemia “APL”,acute monoblastic leukemia, acute erythroleukemic leukemia, acutemegakaryoblastic leukemia, acute myelomonocytic leukemia, acutenonlymphocyctic leukemia, acute undifferentiated leukemia, chronicmyelocytic leukemia “CIVIL”, chronic lymphocytic leukemia “CLL”, hairycell leukemia, multiple myeloma, acute and chronic leukemias, e.g.,lymphoblastic myelogenous and lymphocytic myelocytic leukemias; and (3)lymphomas such as Hodgkin's disease, non-Hodgkin's Lymphoma, Multiplemyeloma, Waldenstrom's macroglobulinemia, Heavy chain disease, andPolycythemia vera.

In another embodiment the conjugates can be administered in vitro, invivo and/or ex vivo to treat patients and/or to modulate the growth ofselected cell populations in patients having anal, astrocytoma,leukemia, lymphoma, head and neck, liver, testicular, cervical, sarcoma,hemangioma, esophageal, eye, laryngeal, mouth, mesothelioma, skin,myeloma, oral, rectal, throat, bladder, breast, uterus, ovary, prostate,lung, colon, pancreas, renal, or gastric cancer.

In another embodiment, the cancers are selected from the groupconsisting of breast cancer, gastric cancer, non-small cell lung cancer(NSCLC), prostate cancer and ovarian cancer.

In another embodiment, the conjugates can be administered in vitro, invivo and/or ex vivo to treat, prevent, reduce the risk of developingand/or delay onset of certain pathologies or disorders, for example, acancer. For example, the conjugates of the disclosure are useful intreating, preventing, delaying the progression of or otherwiseameliorating a symptom of a cancer selected from the group consisting ofanal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer,liver cancer, testicular cancer, cervical cancer, sarcoma, hemangioma,esophageal cancer, eye cancer, laryngeal cancer, mouth cancer,mesothelioma, skin cancer, myeloma, oral cancer, rectal cancer, throatcancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer,prostate cancer, lung cancer, non-small cell lung cancer (NSCLC), coloncancer, pancreatic cancer, renal cancer, and gastric cancer.

In another embodiment the conjugates can be administered in vitro, invivo and/or ex vivo to treat autoimmune diseases, such as systemiclupus, rheumatoid arthritis, psoriasis, and multiple sclerosis; graftrejections, such as renal transplant rejection, liver transplantrejection, lung transplant rejection, cardiac transplant rejection, andbone marrow transplant rejection; graft versus host disease; viralinfections, such as CMV infection, HIV infection, and AIDS; and parasiteinfections, such as giardiasis, amoebiasis, schistosomiasis, and thelike.

In certain embodiments the conjugates can also be used for themanufacture of a medicament useful for treating or lessening theseverity of disorders, such as, characterized by abnormal growth ofcells (e.g., cancer).

In certain embodiments, the therapeutic agent is locally delivered to aspecific target cell, tissue, or organ.

In certain embodiments, in practicing the method of the disclosure, theconjugate further comprises or is associated with a diagnostic label. Incertain exemplary embodiments, the diagnostic label is selected from thegroup consisting of: radiopharmaceutical or radioactive isotopes forgamma scintigraphy and PET, contrast agent for Magnetic ResonanceImaging (MRI), contrast agent for computed tomography, contrast agentfor X-ray imaging method, agent for ultrasound diagnostic method, agentfor neutron activation, moiety which can reflect, scatter or affectX-rays, ultrasounds, radiowaves and microwaves and fluorophores. Incertain exemplary embodiments, the conjugate is further monitored invivo.

Examples of diagnostic labels include, but are not limited to,diagnostic radiopharmaceutical or radioactive isotopes for gammascintigraphy and PET, contrast agent for Magnetic Resonance Imaging(MRI) (for example paramagnetic atoms and superparamagneticnanocrystals), contrast agent for computed tomography, contrast agentfor X-ray imaging method, agent for ultrasound diagnostic method, agentfor neutron activation, and moiety which can reflect, scatter or affectX-rays, ultrasounds, radiowaves and microwaves, fluorophores in variousoptical procedures, etc. Diagnostic radiopharmaceuticals include□-emitting radionuclides, e.g., indium-111, technetium-99m andiodine-131, etc. Contrast agents for MM (Magnetic Resonance Imaging)include magnetic compounds, e.g., paramagnetic ions, iron, manganese,gadolinium, lanthanides, organic paramagnetic moieties andsuperparamagnetic, ferromagnetic and antiferromagnetic compounds, e.g.,iron oxide colloids, ferrite colloids, etc. Contrast agents for computedtomography and other X-ray based imaging methods include compoundsabsorbing X-rays, e.g., iodine, barium, etc. Contrast agents forultrasound based methods include compounds which can absorb, reflect andscatter ultrasound waves, e.g., emulsions, crystals, gas bubbles, etc.Still other examples include substances useful for neutron activation,such as boron and gadolinium. Further, labels can be employed which canreflect, refract, scatter, or otherwise affect X-rays, ultrasound,radiowaves, microwaves and other rays useful in diagnostic procedures.Fluorescent labels can be used for photoimaging. In certain embodimentsa modifier comprises a paramagnetic ion or group.

In another aspect, the disclosure provides a method of treating adisease or disorder in a subject, comprising preparing an aqueousformulation of at least one conjugate of the disclosure and parenterallyinjecting said formulation in the subject.

In another aspect, the disclosure provides a method of treating adisease or disorder in a subject, comprising preparing an implantcomprising at least one conjugate of the disclosure, and implanting saidimplant into the subject. In certain exemplary embodiments, the implantis a biodegradable gel matrix.

In another aspect, the disclosure provides a method for treating of ananimal in need thereof, comprising administering a conjugate accordingto the methods described above.

In another aspect, the disclosure provides a method for eliciting animmune response in an animal, comprising administering a conjugate as inthe methods described above.

In another aspect, the disclosure provides a method of diagnosing adisease in an animal, comprising steps of:

administering a conjugate as in the methods described above, whereinsaid conjugate comprises a detectable molecule; and

detecting the detectable molecule.

In certain exemplary embodiments, the step of detecting the detectablemolecule is performed non-invasively. In certain exemplary embodiments,the step of detecting the detectable molecule is performed usingsuitable imaging equipment.

In one embodiment, a method for treating an animal comprisesadministering to the animal a biodegradable biocompatible conjugate ofthe disclosure as a packing for a surgical wound from which a tumor orgrowth has been removed. The biodegradable biocompatible conjugatepacking will replace the tumor site during recovery and degrade anddissipate as the wound heals.

In certain embodiments, the conjugate is associated with a diagnosticlabel for in vivo monitoring.

The conjugates described above can be used for therapeutic,preventative, and analytical (diagnostic) treatment of animals. Theconjugates are intended, generally, for parenteral administration, butin some cases may be administered by other routes.

In one embodiment, soluble or colloidal conjugates are administeredintravenously. In another embodiment, soluble or colloidal conjugatesare administered via local (e.g., subcutaneous, intramuscular)injection. In another embodiment, solid conjugates (e.g., particles,implants, drug delivery systems) are administered via implantation orinjection.

In another embodiment, conjugates comprising a detectable label areadministered to study the patterns and dynamics of label distribution inanimal body.

In certain embodiments, any one or more of the conjugates disclosedherein may be used in practicing any of the methods described herein.

The pharmaceutical compositions of the conjugates described herein canbe included in a container, pack, or dispenser together withinstructions for administration.

In certain embodiments, the compositions can also contain more than oneactive compound as necessary for the particular indication beingtreated, preferably those with complementary activities that do notadversely affect each other. Alternatively, or in addition, thecomposition can comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

In one embodiment, the active compounds (e.g., conjugates or drugs ofthe disclosure) are administered in combination therapy, i.e., combinedwith other agents, e.g., therapeutic agents, that are useful fortreating pathological conditions or disorders, such as various forms ofcancer, autoimmune disorders and inflammatory diseases. The term “incombination” in this context means that the agents are givensubstantially contemporaneously, either simultaneously or sequentially.If given sequentially, at the onset of administration of the secondcompound, the first of the two compounds is preferably still detectableat effective concentrations at the site of treatment.

For example, the combination therapy can include one or more conjugatesdisclosed herein coformulated with, and/or coadministered with, one ormore additional antibodies, which can be the same as the antibody usedto form the conjugate or a different antibody.

For example, the combination therapy can include one or more therapeuticagent and/or adjuvant. In certain embodiments, the additionaltherapeutic agent is a small molecule inhibitor, another antibody-basedtherapy, a polypeptide or peptide-based therapy, a nucleic acid-basedtherapy and/or other biologic.

In certain embodiments, the additional therapeutic agent is a cytotoxicagent, a chemotherapeutic agent, a growth inhibitory agent, anangiogenesis inhibitor, a PARP (poly(ADP)-ribose polymerase) inhibitor,an alkylating agent, an anti-metabolite, an anti-microtubule agent, atopoisomerase inhibitor, a cytotoxic antibiotic, any other nucleic aciddamaging agent or an immune checkpoint inhibitor. In one embodiment, thetherapeutic agent used in the treatment of cancer, includes but is notlimited to, a platinum compound (e.g., cisplatin or carboplatin); ataxane (e.g., paclitaxel or docetaxel); a topoisomerase inhibitor (e.g.,irinotecan or topotecan); an anthracycline (e.g., doxorubicin(ADRIAMYCIN®) or liposomal doxorubicin (DOXIL®)); an anti-metabolite(e.g., gemcitabine, pemetrexed); cyclophosphamide; vinorelbine(NAVELBINE®); hexamethylmelamine; ifosfamide; etoposide; an angiogenesisinhibitor (e.g., Bevacizumab (Avastin®)), thalidomide, TNP-470, plateletfactor 4, interferon or endostatin); a PARP inhibitor (e.g., Olaparib(Lynparza™)); an immune checkpoint inhibitor, such as for example, amonoclonal antibody that targets either PD-1 or PD-L ((Pembrolizumab(Keytruda®), atezolizumab (MPDL3280A) or Nivolumab (Opdivo®)) or CTA-4(Ipilimumab (Yervoy®), a kinase inhibitor (e.g., sorafenib orerlotinib), a proteasome inhibitor (e.g., bortezomib or carfilzomib), animmune modulating agent (e.g., lenalidomide or IL-2), a radiation agent,an ALK inhibitor (e.g. crizotinib (Xalkori), ceritinib (Zykadia),alectinib (Alecensa), dalantercept (ACE-041), brigatinib (AP26113),entrectinib (NMS-E628), PF-06463922 TSR-011, CEP-37440 and X-396) and/ora biosimilar thereof and/or combinations thereof. Other suitable agentsinclude an agent considered standard of care by those skilled in the artand/or a chemotherapeutic agent well known to those skilled in the art.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofCTLA-4. In some embodiments, the immune checkpoint inhibitor is anantibody against CTLA-4. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against CTLA-4. In other embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstCTLA-4. In one embodiment, the anti-CTLA-4 antibody blocks the bindingof CTLA-4 to CD80 (B7-1) and/or CD86 (B7-2) expressed on antigenpresenting cells. Exemplary antibodies against CTLA-4 include, but arenot limited to, Bristol Meyers Squibb's anti-CTLA-4 antibody ipilimumab(also known as Yervoy®, MDX-010, BMS-734016 and MDX-101); anti-CTLA4Antibody, clone 9H10 from Millipore; Pfizer's tremelimumab (CP-675,206,ticilimumab); and anti-CTLA4 antibody clone BNI3 from Abcam.

In some embodiments, the anti-CTLA-4 antibody is an anti-CTLA-4 antibodydisclosed in any of the following patent publications (hereinincorporated by reference): WO 2001014424; WO 2004035607;US2005/0201994; EP 1212422 B1; WO2003086459; WO2012120125; WO2000037504;WO2009100140; WO200609649; WO2005092380; WO2007123737; WO2006029219;WO20100979597; WO200612168; and WO1997020574. Additional CTLA-4antibodies are described in U.S. Pat. Nos. 5,811,097, 5,855,887,6,051,227, and 6,984,720; in PCT Publication Nos. WO 01/14424 and WO00/37504; and in U.S. Publication Nos. 2002/0039581 and 2002/086014;and/or U.S. Pat. Nos. 5,977,318, 6,682,736, 7, 109,003, and 7,132,281,incorporated herein by reference). In some embodiments, the anti-CTLA-4antibody is for example, those disclosed in: WO 98/42752; U.S. Pat. Nos.6,682,736 and 6,207,156; Hurwitz et al, Proc. Natl. Acad. Sci. USA,95(17): 10067-10071 (1998); Camacho et al, J. Clin. Oncol., 22(145):Abstract No. 2505 (2004) (antibody CP-675206); Mokyr et al, Cancer Res.,58:5301-5304 (1998) (incorporated herein by reference).

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 ligand asdisclosed in WO1996040915.

In some embodiments, the CTLA-4 inhibitor is a nucleic acid inhibitor ofCTLA-4 expression. For example, anti-CTLA4 RNAi molecules may take theform of the molecules described by Mello and Fire in PCT PublicationNos. WO 1999/032619 and WO 2001/029058; U.S. Publication Nos.2003/0051263, 2003/0055020, 2003/0056235, 2004/265839, 2005/0100913,2006/0024798, 2008/0050342, 2008/0081373, 2008/0248576, and 2008/055443;and/or U.S. Pat. Nos. 6,506,559, 7,282,564, 7,538,095, and 7,560,438(incorporated herein by reference). In some instances, the anti-CTLA4RNAi molecules take the form of double stranded RNAi molecules describedby Tuschl in European Patent No. EP 1309726 (incorporated herein byreference). In some instances, the anti-CTLA4 RNAi molecules take theform of double stranded RNAi molecules described by Tuschl in U.S. Pat.Nos. 7,056,704 and 7,078,196 (incorporated herein by reference). In someembodiments, the CTLA4 inhibitor is an aptamer described in PCTPublication No. WO2004081021.

Additionally, the anti-CTLA4 RNAi molecules of the present disclosuremay take the form be RNA molecules described by Crooke in U.S. Pat. Nos.5,898,031, 6,107,094, 7,432,249, and 7,432,250, and European ApplicationNo. EP 0928290 (incorporated herein by reference).

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-L1. In some embodiments, the immune checkpoint inhibitor is anantibody against PD-L1. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against PD-L1. In other or additionalembodiments, the immune checkpoint inhibitor is a human or humanizedantibody against PD-L1. In one embodiment, the immune checkpointinhibitor reduces the expression or activity of one or more immunecheckpoint proteins, such as PD-L1. In another embodiment, the immunecheckpoint inhibitor reduces the interaction between PD-1 and PD-L1.Exemplary immune checkpoint inhibitors include antibodies (e.g., ananti-PD-L1 antibody), RNAi molecules (e.g., anti-PD-L1 RNAi), antisensemolecules (e.g., an anti-PD-L1 antisense RNA), dominant negativeproteins (e.g., a dominant negative PD-L1 protein), and small moleculeinhibitors. Antibodies include monoclonal antibodies, humanizedantibodies, deimmunized antibodies, and Ig fusion proteins. An exemplaryanti-PD-L1 antibody includes clone EH12. Exemplary antibodies againstPD-L1 include: Genentech's MPDL3280A (RG7446); Anti-mouse PD-L1 antibodyClone 10F.9G2 (Cat #BE0101) from BioXcell; anti-PD-L1 monoclonalantibody MDX-1105 (BMS-936559) and BMS-935559 from Bristol-Meyer'sSquibb; MSB0010718C; mouse anti-PD-L1 Clone 29E.2A3; and AstraZeneca'sMEDI4736. In some embodiments, the anti-PD-L1 antibody is an anti-PD-L1antibody disclosed in any of the following patent publications (hereinincorporated by reference): WO2013079174; CN101104640; WO2010036959;WO2013056716; WO2007005874; WO2010089411; WO2010077634; WO2004004771;WO2006133396; WO201309906; US 20140294898; WO2013181634 or WO2012145493.

In some embodiments, the PD-L1 inhibitor is a nucleic acid inhibitor ofPD-L1 expression. In some embodiments, the PD-L1 inhibitor is disclosedin one of the following patent publications (incorporated herein byreference): WO2011127180 or WO2011000841. In some embodiments, the PD-L1inhibitor is rapamycin.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-L2. In some embodiments, the immune checkpoint inhibitor is anantibody against PD-L2. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against PD-L2. In other or additionalembodiments, the immune checkpoint inhibitor is a human or humanizedantibody against PD-L2. In some embodiments, the immune checkpointinhibitor reduces the expression or activity of one or more immunecheckpoint proteins, such as PD-L2. In other embodiments, the immunecheckpoint inhibitor reduces the interaction between PD-1 and PD-L2.Exemplary immune checkpoint inhibitors include antibodies (e.g., ananti-PD-L2 antibody), RNAi molecules (e.g., an anti-PD-L2 RNAi),antisense molecules (e.g., an anti-PD-L2 antisense RNA), dominantnegative proteins (e.g., a dominant negative PD-L2 protein), and smallmolecule inhibitors. Antibodies include monoclonal antibodies, humanizedantibodies, deimmunized antibodies, and Ig fusion proteins.

In some embodiments, the PD-L2 inhibitor is GlaxoSmithKline's AMP-224(Amplimmune). In some embodiments, the PD-L2 inhibitor is rHIgM12B7.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-L1. In some embodiments, the immune checkpoint inhibitor is anantibody against PD-1. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against PD-1. In other embodiments,the immune checkpoint inhibitor is a human or humanized antibody againstPD-1. For example, the inhibitors of PD-1 biological activity (or itsligands) disclosed in U.S. Pat. Nos. 7,029,674; 6,808,710; or U.S.Patent Application Nos: 20050250106 and 20050159351 can be used in thecombinations provided herein. Exemplary antibodies against PD-1 include:Anti-mouse PD-1 antibody Clone J43 (Cat #BE0033-2) from BioXcell;Anti-mouse PD-1 antibody Clone RMP1-14 (Cat #BE0146) from BioXcell;mouse anti-PD-1 antibody Clone EH12; Merck's MK-3475 anti-mouse PD-1antibody (Keytruda®, pembrolizumab, lambrolizumab, h409A1 1); andAnaptysBio's anti-PD-1 antibody, known as ANB011; antibody MDX-1 106(ONO-4538); Bristol-Myers Squibb's human IgG4 monoclonal antibodynivolumab (Opdivo®, BMS-936558, MDX1106); AstraZeneca's AMP-514, andAMP-224; and Pidilizumab (CT-011 or hBAT-1), CureTech Ltd.

Additional exemplary anti-PD-1 antibodies are described by Goldberg etal, Blood 1 10(1): 186-192 (2007), Thompson et al, Clin. Cancer Res.13(6): 1757-1761 (2007), and Korman et al, International Application No.PCT/JP2006/309606 (publication no. WO 2006/121168 A1), each of which areexpressly incorporated by reference herein. In some embodiments, theanti-PD-1 antibody is an anti-PD-1 antibody disclosed in any of thefollowing patent publications (herein incorporated by reference):WO014557; WO2011110604; WO2008156712; US2012023752; WO2011110621;WO2004072286; WO2004056875; WO20100036959; WO2010029434; WO201213548;WO2002078731; WO2012145493; WO2010089411; WO2001014557; WO2013022091;WO2013019906; WO2003011911; US20140294898; and WO2010001617.

In some embodiments, the PD-1 inhibitor is a PD-1 binding protein asdisclosed in WO200914335 (herein incorporated by reference).

In some embodiments, the PD-1 inhibitor is a peptidomimetic inhibitor ofPD-1 as disclosed in WO2013132317 (herein incorporated by reference).

In some embodiments, the PD-1 inhibitor is an anti-mouse PD-1 mAb: cloneJ43, BioXCell (West Lebanon, N.H.).

In some embodiments, the PD-1 inhibitor is a PD-L1 protein, a PD-L2protein, or fragments, as well as antibody MDX-1 106 (ONO-4538) testedin clinical studies for the treatment of certain malignancies (Brahmeret al., J Clin Oncol. 2010 28(19): 3167-75, Epub 2010 Jun. 1). Otherblocking antibodies may be readily identified and prepared by theskilled person based on the known domain of interaction between PD-1 andPD-L1/PD-L2, as discussed above. For example, a peptide corresponding tothe IgV region of PD-1 or PD-L1/PD-L2 (or to a portion of this region)could be used as an antigen to develop blocking antibodies using methodswell known in the art.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofIDO1. In some embodiments, the immune checkpoint inhibitor is a smallmolecule against IDO1. Exemplary small molecules against IDO1 include:Incyte's INCB024360, NSC-721782 (also known as 1-methyl-D-tryptophan),and Bristol Meyers Squibb's F001287.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofLAG3 (CD223). In some embodiments, the immune checkpoint inhibitor is anantibody against LAG3. In some embodiments, the immune checkpointinhibitor is a monoclonal antibody against LAG3. In other or additionalembodiments, the immune checkpoint inhibitor is a human or humanizedantibody against LAG3. In additional embodiments, an antibody againstLAG3 blocks the interaction of LAG3 with major histocompatibilitycomplex (MHC) class II molecules. Exemplary antibodies against LAG3include: anti-Lag-3 antibody clone eBioC9B7W (C9B7W) from eBioscience;anti-Lag3 antibody LS-B2237 from LifeSpan Biosciences; IMP321 (ImmuFact)from Immutep; anti-Lag3 antibody BMS-986016; and the LAG-3 chimericantibody A9H12. In some embodiments, the anti-LAG3 antibody is ananti-LAG3 antibody disclosed in any of the following patent publications(herein incorporated by reference): WO2010019570; WO2008132601; orWO2004078928.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst TIM3 (also known as HAVCR2). In some embodiments, the immunecheckpoint inhibitor is a monoclonal antibody against TIM3. In other oradditional embodiments, the immune checkpoint inhibitor is a human orhumanized antibody against TIM3. In additional embodiments, an antibodyagainst TIM3 blocks the interaction of TIM3 with galectin-9 (Ga19). Insome embodiments, the anti-TIM3 antibody is an anti-TIM3 antibodydisclosed in any of the following patent publications (hereinincorporated by reference): WO2013006490; WO201155607; WO2011159877; orWO200117057. In another embodiment, a TIM3 inhibitor is a TIM3 inhibitordisclosed in WO2009052623.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst B7-H3. In one embodiment, the immune checkpoint inhibitor isMGA271.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst MR. In one embodiment, the immune checkpoint inhibitor isLirilumab (IPH2101). In some embodiments, an antibody against MR blocksthe interaction of KIR with HLA.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD137 (also known as 4-1BB or TNFRSF9). In one embodiment, theimmune checkpoint inhibitor is urelumab (BMS-663513, Bristol-MyersSquibb), PF-05082566 (anti-4-1BB, PF-2566, Pfizer), or XmAb-5592(Xencor). In one embodiment, an anti-CD137 antibody is an antibodydisclosed in U.S. Published Application No. US 2005/0095244; an antibodydisclosed in issued U.S. Pat. No. 7,288,638 (such as 20H4.9-IgG4 [1007or BMS-663513] or 20H4.9-IgG1 [BMS-663031]); an antibody disclosed inissued U.S. Pat. No. 6,887,673 [4E9 or BMS-554271]; an antibodydisclosed in issued U.S. Pat. No. 7,214,493; an antibody disclosed inissued U.S. Pat. No. 6,303,121; an antibody disclosed in issued U.S.Pat. No. 6,569,997; an antibody disclosed in issued U.S. Pat. No.6,905,685; an antibody disclosed in issued U.S. Pat. No. 6,355,476; anantibody disclosed in issued U.S. Pat. No. 6,362,325 [1D8 or BMS-469492;3H3 or BMS-469497; or 3E1]; an antibody disclosed in issued U.S. Pat.No. 6,974,863 (such as 53A2); or an antibody disclosed in issued U.S.Pat. No. 6,210,669 (such as 1D8, 3B8, or 3E1). In a further embodiment,the immune checkpoint inhibitor is one disclosed in WO 2014036412. Inanother embodiment, an antibody against CD137 blocks the interaction ofCD137 with CD137L.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst PS. In one embodiment, the immune checkpoint inhibitor isBavituximab.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD52. In one embodiment, the immune checkpoint inhibitor isalemtuzumab.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD30. In one embodiment, the immune checkpoint inhibitor isbrentuximab vedotin. In another embodiment, an antibody against CD30blocks the interaction of CD30 with CD30L.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD33. In one embodiment, the immune checkpoint inhibitor isgemtuzumab ozogamicin.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD20. In one embodiment, the immune checkpoint inhibitor isibritumomab tiuxetan. In another embodiment, the immune checkpointinhibitor is ofatumumab. In another embodiment, the immune checkpointinhibitor is rituximab. In another embodiment, the immune checkpointinhibitor is tositumomab.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst CD27 (also known as TNFRSF7). In one embodiment, the immunecheckpoint inhibitor is CDX-1127 (Celldex Therapeutics). In anotherembodiment, an antibody against CD27 blocks the interaction of CD27 withCD70.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst OX40 (also known as TNFRSF4 or CD134). In one embodiment, theimmune checkpoint inhibitor is anti-OX40 mouse IgG. In anotherembodiment, an antibody against Ox40 blocks the interaction of OX40 withOX40L.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst glucocorticoid-induced tumor necrosis factor receptor (GITR). Inone embodiment, the immune checkpoint inhibitor is TRX518 (GITR, Inc.).In another embodiment, an antibody against GITR blocks the interactionof GITR with GITRL.

In some embodiments, the immune checkpoint inhibitor is an antibodyagainst inducible T-cell COStimulator (ICOS, also known as CD278). Inone embodiment, the immune checkpoint inhibitor is MEDI570 (MedImmune,LLC) or AMG557 (Amgen). In another embodiment, an antibody against ICOSblocks the interaction of ICOS with ICOSL and/or B7-H2.

In some embodiments, the immune checkpoint inhibitor is an inhibitoragainst BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2,or SLAM. As described elsewhere herein, an immune checkpoint inhibitorcan be one or more binding proteins, antibodies (or fragments orvariants thereof) that bind to immune checkpoint molecules, nucleicacids that downregulate expression of the immune checkpoint molecules,or any other molecules that bind to immune checkpoint molecules (i.e.small organic molecules, peptidomimetics, aptamers, etc.). In someinstances, an inhibitor of BTLA (CD272) is HVEM. In some instances, aninhibitor of CD160 is HVEM. In some cases, an inhibitor of 2B4 is CD48.In some instances, an inhibitor of LAIR1 is collagen. In some instances,an inhibitor of TIGHT is CD112, CD113, or CD155. In some instances, aninhibitor of CD28 is CD80 or CD86. In some instances, an inhibitor ofLIGHT is HVEM. In some instances, an inhibitor of DR3 is TL1A. In someinstances, an inhibitor of CD226 is CD155 or CD112. In some cases, aninhibitor of CD2 is CD48 or CD58. In some cases, SLAM is self inhibitoryand an inhibitor of SLAM is SLAM.

In certain embodiments, the immune checkpoint inhibitor inhibits acheckpoint protein that include, but are not limited to CTLA4 (cytotoxicT-lymphocyte antigen 4, also known as CD152), PD-L1 (programmed celldeath 1 ligand 1, also known as CD274), PDL2 programmed cell deathprotein 2), PD-1 (programmed cell death protein 1, also known as CD279),a B-7 family ligand (B7-H1, B7-H3, B7-H4) BTLA (B and T lymphocyteattenuator, also known as CD272), HVEM, TIM3 (T-cell membrane protein3), GAL9, LAG-3 (lymphocyte activation gene-3; CD223), VISTA, KIR(killer immunoglobulin receptor), 2B4 (also known as CD244), CD160,CGEN-15049, CHK1 (Checkpoint kinase 1), CHK2 (Checkpoint kinase 2), A2aR(adenosine A2a receptor), CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86,CD137, CD226, CD276, DR3, GITR, HAVCR2, HVEM, IDO1 (indoleamine2,3-dioxygenase 1), IDO2 (indoleamine 2,3-dioxygenase 2), ICOS(inducible T cell costimulator), LAIR1, LIGHT (also known as TNFSF14, aTNF family member), MARCO (macrophage receptor with collagenousstructure), OX40 (also known as tumor necrosis factor receptorsuperfamily, member 4, TNFRSF4, and CD134) and its ligand OX40L (CD252),SLAM, TIGHT, VTCN1 or a combination thereof.

In certain embodiments, the immune checkpoint inhibitor interacts with aligand of a checkpoint protein that comprises CTLA-4, PDL1, PDL2, PD1,BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1,CHK2, A2aR, a B-7 family ligand, CD2, CD27, CD28, CD30, CD40, CD70,CD80, CD86, CD137, CD226, CD276, DR3, GITR, HAVCR2, HVEM, IDO1, IDO2,ICOS (inducible T cell costimulator), LAIR1, LIGHT, MARCO (macrophagereceptor with collagenous structure), OX-40, SLAM, TIGHT, VTCN1 or acombination thereof.

In certain embodiments, the immune checkpoint inhibitor inhibits acheckpoint protein that comprises CTLA-4, PDL1, PD1 or a combinationthereof.

In certain embodiments, the immune checkpoint inhibitor inhibits acheckpoint protein that comprises CTLA-4 and PD1 or a combinationthereof.

In certain embodiments, the immune checkpoint inhibitor comprisespembrolizumab (MK-3475), nivolumab (BMS-936558), pidilizumab (CT-011),AMP-224, MDX-1 105, durvalumab (MEDI4736), MPDL3280A, BMS-936559,IPH2101, TSR-042, TSR-022, ipilimumab, lirilumab, atezolizumab,avelumab, tremelimumab, or a combination thereof.

In certain embodiments, the immune checkpoint inhibitor is nivolumab(BMS-936558), ipilimumab, pembrolizumab, atezolizumab, tremelimumab,durvalumab, avelumab, or a combination thereof.

In certain embodiments, the immune checkpoint inhibitor ispembrolizumab.

Throughout the description, where compounds, scaffolds, and compositionsare described as having, including, or comprising specific components,it is contemplated that compositions also consist essentially of, orconsist of, the recited components. Similarly, where methods orprocesses are described as having, including, or comprising specificprocess steps, the processes also consist essentially of, or consist of,the recited processing steps. Further, it should be understood that theorder of steps or order for performing certain actions is immaterial solong as the invention remains operable. Moreover, two or more steps oractions can be conducted simultaneously.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illustrate theinvention and is not to be construed as a limitation on the scope of theclaims unless explicitly otherwise claimed. No language in thespecification is to be construed as indicating that any non-claimedelement is essential to what is claimed.

Synthetic Methods

Any available techniques can be used to make the conjugates orcompositions thereof, and intermediates and components (e.g., scaffolds)useful for making them. For example, semi-synthetic and fully syntheticmethods may be used.

The general methods of producing the conjugates or scaffolds disclosedherein are illustrated in Schemes 1 and 2 below. More specific methodsof syntheses are described in the Examples. The variables (e.g., M^(P),M^(A), L³, W^(D), W^(M), L^(D), and L^(P′), etc.) in these schemes havethe same definitions as described herein unless otherwise specified.

The synthetic processes of the disclosure can tolerate a wide variety offunctional groups; therefore various substituted starting materials canbe used. The processes generally provide the desired final compound ator near the end of the overall process, although it may be desirable incertain instances to further convert the compound to a pharmaceuticallyacceptable salt, ester or prodrug thereof.

Drug compounds used for the conjugates of the present disclosure can beprepared in a variety of ways using commercially available startingmaterials, compounds known in the literature, or from readily preparedintermediates, by employing standard synthetic methods and procedureseither known to those skilled in the art, or which will be apparent tothe skilled artisan in light of the teachings herein. Standard syntheticmethods and procedures for the preparation of organic molecules andfunctional group transformations and manipulations can be obtained fromthe relevant scientific literature or from standard textbooks in thefield. Although not limited to any one or several sources, classic textssuch as Smith, M. B., March, J., March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, 5^(th) edition, John Wiley & Sons:New York, 2001; and Greene, T. W., Wuts, P. G. M., Protective Groups inOrganic Synthesis, 3^(rd) edition, John Wiley & Sons: New York, 1999,incorporated by reference herein, are useful and recognized referencetextbooks of organic synthesis known to those in the art. The followingdescriptions of synthetic methods are designed to illustrate, but not tolimit, general procedures for the preparation of compounds of thepresent disclosure.

Conjugates of the present disclosure and the drug compounds includedtherein can be conveniently prepared by a variety of methods familiar tothose skilled in the art. The conjugates or compounds of the disclosurewith each of the formulae described herein may be prepared according tothe following procedures from commercially available starting materialsor starting materials which can be prepared using literature procedures.These procedures show the preparation of representative conjugates ofthis disclosure.

Conjugates designed, selected and/or optimized by methods describedabove, once produced, can be characterized using a variety of assaysknown to those skilled in the art to determine whether the conjugateshave biological activity. For example, the conjugates can becharacterized by conventional assays, including but not limited to thoseassays described below, to determine whether they have a predictedactivity, binding activity and/or binding specificity.

Furthermore, high-throughput screening can be used to speed up analysisusing such assays. As a result, it can be possible to rapidly screen theconjugate molecules described herein for activity, using techniquesknown in the art. General methodologies for performing high-throughputscreening are described, for example, in Devlin (1998) High ThroughputScreening, Marcel Dekker; and U.S. Pat. No. 5,763,263. High-throughputassays can use one or more different assay techniques including, but notlimited to, those described below.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES

The following working examples are illustrative of the linkers, drugmolecules and antibodies or antibody fragments, and methods forpreparing same. These are not intended to be limiting and it will bereadily understood by one of skill in the art that other reagents ormethods may be utilized.

Abbreviations

The following abbreviations are used in the reaction schemes andsynthetic examples, which follow. This list is not meant to be anall-inclusive list of abbreviations used in the application asadditional standard abbreviations, which are readily understood by thoseskilled in the art of organic synthesis, can also be used in thesynthetic schemes and examples Abbreviations:

-   -   ACN Acetonitrile    -   Alloc Allyloxycarbonyl    -   AcOH Acetic acid    -   BOC tert-butyloxycarbonyl    -   CDI Bis(1H-imidazol-1-yl)methanone    -   EDTA Ethylenediaminetetraacetic acid    -   DCM Dichloromethane    -   DIEA N,N-Diisopropylethylamine    -   DMAP N,N-Dimethylpyridin-4-amine    -   DMF Dimethylformamide    -   EDC.HCl 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide        hydrochloride    -   HATU        1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium        3-oxid hexafluorophosphate)    -   HOAt 1-Hydroxy-7-azabenzotriazole    -   HOBt Hydroxybenzotriazole    -   NHS 1-Hydroxypyrrolidine-2,5-dione (i.e., N-hydroxy-succinimide)    -   TEAA Triethylammonium acetate    -   TCEP Tris[2-carboxyethyl] phosphine    -   THF Tetrahydrofuran    -   MI Maleimide or maleimido    -   PDI Polydispersity index    -   RP-HPLC Reverse-phase high performance liquid chromatography    -   SEC Size exclusion chromatography    -   WCX Weak cation exchange chromatography        General Information

8,8-bis((2-carboxyethoxy)methyl)-3,6-dioxo-1-phenyl-2,10-dioxa-4,7-diazadodecane-12-carboxylicacid was purchased from eNovation Chemicals LLC, Bridgewater, N.J.

2,5-dioxopyrrolidin-1-yl3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1yl)propanamido)ethoxy)ethoxy)propanoatewas purchased from QuantaBiodesign Ltd,

Boc-Glu(OtBu)-OH, di-tert-butyl dicarbonate, (S)-benzyl2-((tert-butoxycarbonyl)amino)-3-hydroxypropanoate,3-methoxy-3-oxopropan-1-ammonium chloride, Boc-Asp(OAll)-OH werepurchased from Sigma-Aldrich

2-(2-(2-(2-aminoacetamido)acetamido)acetamido)acetic acid was purchasedfrom Bachem Americas Inc. Torrance, Calif.

H₂N-PEG₁₂-OMe was purchased from Quanta Biodesign, Ltd.

H₂N-PEG₈-OMe was purchased from ChemPep Inc., Wellington, Fla.

H₂N-PEG₆-OMe was purchased from BroadPharm, San Diego, Calif.

Glucamine was purchased from TCI America.

tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate was purchasedfrom A1 BioChem Labs, Wilmington, N.C.

XMT-1535 is disclosed in co-pending application U.S. Ser. No. 15/457,574filed Mar. 13, 2017.

Tumor growth inhibition (% TGI) was defined as the percent difference inmedian tumor volumes (MTVs) between treated and control groups.

Treatment efficacy was determined from the incidence and magnitude ofregression responses of the tumor size observed during the study.Treatment may cause partial regression (PR) or complete regression (CR)of the tumor in an animal. In a PR response, the tumor volume was 50% orless of its Day 1 volume for three consecutive measurements during thecourse of the study, and equal to or greater than 13.5 mm³ for one ormore of these three measurements. In a CR response, the tumor volume wasless than 13.5 mm3 for three consecutive measurements during the courseof the study. An animal with a CR response at the termination of a studywas additionally classified as a tumor-free survivor (TFS). Animals weremonitored for regression responses.

HPLC purification was performed on a Phenomenex Gemini 5 μm 110 Å,250×10 mm, 5 micron, semi-preparation column.

Whenever possible the drug content of the conjugates was determinedquantitatively by chromatography.

The protein content of the protein-polymer-drug conjugates wasdetermined spectrophotometrically at 280 nm or by ELISA.

Antibody-polymer-drug conjugates, drug carrying-polymeric scaffolds, orantibody-carrying polymer scaffolds can be purified (i.e., removal ofresidual unreacted drug, antibody, or polymeric starting materials) byextensive diafiltration. If necessary, additional purification by sizeexclusion chromatography can be conducted to remove any aggregatedantibody-polymer-drug conjugates. In general, the antibody-polymer-drugconjugates as purified typically contain <5% (e.g., <2% w/w) aggregatedantibody -polymer-drug conjugates as determined by SEC; <0.5% (w/w)(e.g., <0.1% w/w) free (unconjugated) drug as determined by RP-HPLC orLC-MS/MS; <1% (w/w) of free polymer-drug conjugate as determined by SECand/or RP-HPLC and <2% (w/w) (e.g., <1% w/w) unconjugated antibody orantibody fragment as determined by HIC-HPLC and/or WCX HPLC. Reduced orpartially reduced antibodies were prepared using procedures described inthe literature, see, for example, Francisco et al., Blood 102 (4):1458-1465 (2003). The total drug (conjugated and unconjugated)concentration was determined by RP-HPLC or back-calculation from DARmeasured by CE-SDS.

RP-HPLC, or CE-SDS were used to characterize the specificity anddistribution of the cysteine bioconjugation sites in thePBRM-polymer-drug conjugates. The results gave the positionaldistribution of the drug-polymer conjugates on the heavy (H) and light(L) chains of the PBRM.

To determine the concentration of the free drug in a biological sample,an acidified sample was treated with acetonitrile. The free drug wasextracted and the acetonitrile supernatant was analyzed. To determinethe concentration of conjugated AF-HPA, the sample was subjected toexhaustive basic hydrolysis followed by immunocapture using anti-IgG1antibody magnetic beads. The acetonitrile supernatant containing thereleased AF-HPA and AF was analyzed RP-HPLC. The total antibody wasmeasured using the unique peptide after digestion.

Analysis of free AF and AF-HPA was conducted by RP-HPLC using a C-4column, an acetonitrile gradient and UV detection. Peak areas areintegrated and compared to AF and AF-HPA standards. The method isquantitive for AF-HPA and AF in plasma and tissue homogenates and linearover the concentration ranges of 0.1 to 150 ng/mL. The total drug(AF-HPA) released after hydrolysis with NaOH was measured under the samecondition with the dynamic range from 1 ng/mL to 5000 ng/mL. The totalantibody standards range from 0.1 μg/mL to 100 μg/mL.

Example 1: Synthesis of Monomeric PEG8 Scaffold (7)

Part A:

Potassium carbonate (2.245 g, 16.25 mmol) was dissolved in dioxane/water(2:1, 120 mL) at room temperature, then2-(2-(2-(2-aminoacetamido)acetamido)acetamido)acetic acid (compound 1, 2g, 8.12 mmol) was added in one portion and the mixture was stirred untilthe reagents dissolved. Then di-tert-butyl dicarbonate (3.55 g, 16.25mmol) in 1,4-dioxane (5 ml) was added dropwise and the reaction mixturewas stirred at room temperature for 25 hours at which point LC-MSanalysis indicated the reaction was complete. The crude mixture wasneutralized to ˜pH 7 with 1 M HCl, then adjusted to pH 2 with citricacid (0.9 equivalents), then concentrated by rotary evaporation andpurified by RP C18 column CombiFlash chromatography to give compound 2(2.57 g, 91% yield). ¹H-NMR (400 MHz, DMSO-d₆): δ8.06-8.20 ppm (m, 2H,2NH), 8.01 (bs, 1H, NH), 6.98 (bs, 1H, NH), 3.73 (s, 6H, 3CH2), 3.5-3.6(d, 2H, CH2), 1.37 (s, 9H, t-Bu). ESI MS: C₁₃H₂₂N₄O₇ [M+H]⁺ 347.16 found347.1.

Part B:

CDI (0.659 g, 4.06 mmol) was added in one portion to (S)-benzyl2-((tert-butoxycarbonyl)amino)-3-hydroxypropanoate (1 g, 3.39 mmol) inTHF (5 mL) at 0° C. The reaction mixture was stirred at room temperaturefor 1 hour at which point LC-MS analysis indicated the reaction wascomplete. Then 3-methoxy-3-oxopropan-1-ammonium chloride (1.182 g, 8.47mmol) and DIEA (1.774 ml, 10.16 mmol) in DMF (3 mL) was added and thestirring continued at room temperature for 3.5 hours at which pointLC-MS indicated the reaction was complete. The crude mixture wasconcentrated, neutralized, purified by RP C18 column CombiFlashchromatography to give Boc-Ser(O-β-Ala-OMe)-OBenzyl (1.085 g, 75%yield). ¹H-NMR (400 MHz, CDCl₃): δ 5.3-5.42 (bs, 1H, NH), 5.1-5.28 (m,3H, OCH₂—Ar), 4.5-4.6 (bs, 1H, CHN), 4.25-4.38 (dd, 2H, CH₂O), 3.71 (s,3H, OMe), 3.35-3.50 (m, CH₂N), 2.53 (t, 2H, CH₂CO), 1.45 (s, 9H, t-Bu).ESI MS: C₂₀H₂₈N₂O₈ [M+Na]⁺ 447.17; found 447.1.

Boc-Ser(β-Ala-OMe)-OBenzyl (1.085 g, 2.56 mmol) was dissolved in DCM(3.3 mL) containing TFA (1.1 mL) at 0° C. for 1 hour and then stirred atroom temperature for 30 minutes until LC-MS indicated the reaction wascomplete. The TFA salt of benzyl2-amino-3-(((3-methoxy-3-oxopropyl)carbamoyl)oxy)propanoate wasconcentrated by rotary evaporation.

Compound 2 (805 mg, 2.324 mmol) was dissolved in DMF (17 mL), cooled to0° C. and NHS (401 mg, 3.49 mmol) was added followed by EDC (668 mg,3.49 mmol) in DMF (6 mL). Separately the TFA salt of benzyl2-amino-3-(((3-methoxy-3-oxopropyl)carbamoyl)oxy)propanoate (829 mg,2.56 mmol) was dissolved in DMF (10 mL), stirred at 0° C. for 10 min andthen DIEA (0.812 ml, 4.65 mmol), was added. This homogeneous mixture wasadded to the reaction mixture containing compound 2. The resultingmixture was stirred at 0° C. for about 1 hour and then at roomtemperature overnight. The reaction mixture was concentrated by rotaryevaporation, pH 5-7, followed by purification by RP HPLC, to givecompound 3 (149 mg, 80% yield). ESI MS: C₂₈H₄₀N₆O₁₂ [M+H]⁺ 653.28, found653.2.

Part C:

Compound 3 (1.213 g, 1.859 mmol) was dissolved in ethanol (80 mL) withheating and stirring, then cooled to room temperature and 10% Pd/C (198mg) was added under H₂. After 2 hours at room temperature LC-MSindicated the reaction was complete. The crude product was filtered andconcentrated by rotary evaporation to give compound 4 (1.103 g, ˜99%yield). ESI MS: C₂₁H₃₄N₆O₁₂ [M+H]⁺ 563.23, found 563.1.

Part D:

Compound 4 (1.05 g, 1.86 mmol) was dissolved in a mixture of toluene/DMFand concentrated by rotary evaporation multiple times to remove theresidual ethanol, then H₂N-PEG₈-OMe (856 mg, 2.231 mmol) in DMF (15 mL)was added. The reaction mixture was cooled to 0° C., then HOAt (380 mg,2.79 mmol), HATU (848 mg, 2.231 mmol), DMF (5 mL) and DIEA (0.812 ml,4.65 mmol) were added. LC-MS showed the reaction was incomplete.Additional HOAt (253 mg) and HATU (353 mg) were added and the mixturewarmed to room temperature. After 5 hours at room temperature thereaction mixture was warmed to 35° C. for ˜45 minutes when LC-MS showedcompletion of the reaction. The crude reaction mixture was concentratedby rotary evaporation and purified by RP C18 column CombiFlashchromatography using ACN/water containing HOAc (0.1%) gradient aseluant, to give compound 5 (1.26 g, 73% yield). ¹H-NMR (400 MHz,DMSO-d₆) δ 8.00-8.25 (m, 5H, NH), 7.20 (t, 1H, NH), 7.00 (t, 1H, NH),4.35-4.55 (m, 1H, CH), 3.93-4.20 (m, 2H, CH₂), 3.65-3.85 (m, 6H, CH₂),3.58 (s, 3H, OMe), 3.55-3.63 (m, 2H, CH₂), 3.50 (bs, 26H, CH₂O),3.35-3.45 (m, 4H, CH₂), 3.23 (s, 3H, OMe), 3.12-3.25 (m, 4H, CH₂N), 2.45(t, 2H, CH₂CO), 1.37 (s, 9H, Ot-Bu). ESI MS: C₃₈H₆₉N₇O₁₉ [M+H]⁺ 928.47,found 928.3.

Part E:

Compound 5 (300 mg, 0.323 mmol) was dissolved in water (3.5 mL) and LiOH(77 μL of 4 M solution) at 0° C. was added. The reaction was monitoredby LC-MS. After 1.5 hours additional aqueous LiOH (38 μL of 4 Msolution) was added. After 20 minutes at 0° C., the reaction wasneutralized with 1 M HCl, pH˜5, followed by purification on C18 RPCombiFlash column using ACN/water containing HOAc (0.1%) gradient aseluant, to give compound 6 (253 mg, 86% yield). ESI MS: C₃₇H₆₇N₇O₁₉[M+H]⁺ 914.45, found 914.0.

Part F:

Compound 6 (117 mg, 0.128 mmol) was dissolved in DCM (3.75 mL) andtreated at 0° C. with TFA (1.25 mL). After 45 min the reaction mixturewas concentrated by rotary evaporation at 0° C. to give the TFA salt.

The TFA-salt of compound 6 (119 mg, 0.128 mmol) was dissolved in DMF(1.7 mL), then DIEA (5 equivalents) was added. The reaction mixture wascooled to 0° C., then2,5-dioxopyrrolidin-1-yl3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1yl)propanamido)ethoxy)ethoxy)propanoate (109 mg, 0.256 mmol) in DMF (0.5 mL) was added.Stirring was continued for 30 minutes at 0° C. and then at roomtemperature for 30 minutes, when LC-MS analysis indicated the reactionwas complete. The reaction mixture was concentrated by rotaryevaporation and purified by RP C18 column CombiFlash chromatographyusing ACN/water containing HOAc (0.1%) gradient as eluant, to givecompound 7 (86 mg, 60% yield).

¹H NMR (400 MHz, DMSO-d₆+20 μL of D₂O) δ 12.07-12.25 (bs, 1H, CO₂H),7.9-8.37 (m, 7H, NH), 7.11 (t, 1H, NH), 6.90-7.05 (s, 2H, CH═CH),4.35-4.57 (m, 1H, CH), 3.95-4.20 (m, 2H, CH₂), 3.65-3.83 (m, 8H, CH₂),3.25-3.65 (m, 40H, CH₂), 3.23 (s, 3H, OMe), 3.07-3.25 (m, 6H, CH₂),2.20-2.43 (m, 6H, CH₂CO).

ESI MS: C₄₆H₇₇N₉O₂₃ [M+H]⁺ 1124.52, found 1124.0.

Example 2: Synthesis of Trimeric PEG8 Scaffold (17)

Part A:

Compound 8 (0.5 g, 0.946 mmol) was dissolved in DCM/DMF (90:10, 25 mL),then HOAt (0.386 g, 2.84 mmol) and EDC (0.635 g, 3.31 mmol) were addedand the resulting mixture was stirred at 0° C. for 15-20 minutes.Separately β-Ala-OMe hydrochloride (0.462 g, 3.31 mmol) in DCM/DMF (9:1,5 mL) was treated with DIEA (0.578 ml, 3.31 mmol) and then added to thereaction mixture. Additional DIEA (0.578 ml) was added and the reactionmixture was allowed to warm up to room temperature and stirredovernight. LC-MS indicated the reaction was nearly complete. The crudereaction mixture was diluted with DCM and washed successively with HCl(0.2 M in brine), brine and aqueous NaOH (0.2M in brine) and brine. Theorganic phase was dried with MgSO₄, filtered and concentrated to givecompound 9 that was used as is. ESI MS: C₃₅H₅₃N₅O₁₅ [M+H]⁺ 784.35; found784.0.

Part B:

To compound 9 (735 mg, 0.938 mmol) in ethanol (50 mL) was added 10% Pd/C(100 mg, 0.094 mmol) under H₂. When LC-MS indicated the reaction wascomplete, the reaction mixture was filtered through Celite andconcentrated to give compound 10 (0.565 g. 93% yield). ESI MS:C₂₇H₄₇N₅O₁₃ [M+H]⁺ 650.32; found 650.0.

Part C:

To tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (1 g, 4.03mmol) in CHCl₃ (25 ml), at 0° C. was added dihydrofuran-2,5-dione (0.403g, 4.03 mmol) and the reaction mixture was allowed to warm up to roomtemperature. After 20 hours, the reaction mixture was concentrated byrotary evaporation to give compound 11 (1.535 g). ESI MS: C₁₅H₂₈N₂O₇[M−H]⁻ 347.19; found 347.2.

Part D:

To compound 11 (201 mg, 0.576 mmol) in DMF (4.3 mL) at 0° C. was addedHOAt) (115 mg, 0.847 mmol) and EDC (162 mg, 0.847 mmol). After 15minutes, compound 10 (220 mg, 0.339 mmol) in DMF (0.7 mL) was addedfollowed by DIEA (0.148 ml, 0.847 mmol). The reaction mixture wasallowed to warm up slowly to room temperature and stirred overnight,then concentrated by rotary evaporation and purified by RP C18 columnCombiFlash chromatography using ACN/water containing HOAc (0.1%)gradient as eluant, to give compound 12 (250 mg, 75% yield). ESI MS:C₄₂H₇₃N₇O₁₉ [M+H]⁺ 979.5; found 980.0.

Part E:

To compound 12 (250 mg, 0.255 mmol) in MeOH (4 ml) and water (0.5 mL) at0° C. was added NaOH (51.0 mg, 1.275 mmol in water (0.5 mL)). After 45minutes the reaction mixture was warmed to room temperature and stirredfor 3 hours at room temperature, then neutralized with aqueous HCl (1M),concentrated and purified by C18 RP chromatography to give compound 13(207 mg, 87% yield. ESI MS: C₃₉H₆₇N₇O₁₉ [M+H]⁺ 938.45; found 938.0.

Part F:

To compound 13 (90 mg, 0.096 mmol) in water (1.5 mL) at 0° C., was addedHOAt (65.3 mg, 0.480 mmol) in NMP (350 μL) followed by EDC (92 mg, 0.480mmol). The reaction mixture was stirred for 15 minutes. Separatelycompound 5 (294 mg, 0.355 mmol) was deprotected using TFA, neutralizedand added to the reaction mixture. The pH of the resulting reactionmixture was adjusted to pH 7 and the reaction mixture was warmed slowlyto room temperature and stirred overnight. The crude material wasconcentrated by rotary evaporation and purified by RP C18 columnCombiFlash chromatography using ACN/water containing HOAc (0.1%)gradient as eluant, to give compound 14 (258 mg, 80% yield). ESI MS:C₁₃₈H₂₄₄N₂₈O₆₇ [M+2H]²⁺/2=1683.3; found 1683.3.

Part G:

Compound 14 (222 mg, 0.066 mmol) in water (3 mL) was reacted with LiOH(16 mg) as described in Example 1, Part E, to give compound 15 (180 mg,82% yield). ESI MS: C₁₃₅H₂₃₈N₂₈O₆₇ [M+2H]²⁺/2=1662.8; found 1662.3.

Part H:

To compound 15 (157 mg, 0.047 mmol) in water (4.5 mL) at 0° C., wasadded HOAt (32.1 mg, 0.236 mmol) in NMP (500 μL) and EDC (27.2 mg, 0.142mmol) and the resulting reaction mixture was stirred for 15 minutes.Separately a mixture of auristatin F-hydroxypropyl amide-alanineTFA-salt (182 mg, 0.165 mmol, prepared as described in U.S. Pat. No.8,865,383) in water (1.5 mL) and NMP (250 μL) was neutralized, and thenadded to the reaction mixture at 0° C. The stirring was continued at 0°C. for 1.5 hours and then stirred at room temperature for 23 hours whenLC-MS showed completion of the reaction. The crude reaction mixture wasconcentrated by rotary evaporation and purified by RP C18 columnCombiFlash chromatography using ACN/water containing HOAc (0.1%)gradient as eluant, to give compound 16 (177 mg, 63% yield). ESI MS:C₂₇₃H₄₆₉N₄₉O₉₁ [M+3H]³⁺/3=1964.46; found 1964.5.

Part I:

The TFA salt of compound 16 (40.3 mg, 6.96 μmol) in DCM was treated withDIEA until the reaction mixture was basic, then cooled to 0° C. beforethe addition of2,5-dioxopyrrolidin-1-yl3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1yl)propanamido)ethoxy)ethoxy)propanoate (14.80 mg, 34.8 μmol). After 45 minutes LC-MS analysisindicated the reaction was complete The crude reaction mixture wasconcentrated by rotary evaporation and purified by RP C18 columnCombiFlash chromatography using ACN/water containing HOAc (0.1%)gradient as eluant, to give scaffold 17 (30 mg, 71% yield). ESI MS:C₂₈₂H₄₇₉N₅₁O₉₅ [M+5H]⁵⁺/5=1221.084 found 1221.0; [M+7H]⁷⁺/7=872.49 found872.4.

Example 3: Synthesis of Compound 18

To 2,2-dimethyl-4,9-dioxo-3,11-dioxa-5,8-diazatridecan-13-oic acid (4.60mg, 0.017 mmol, prepared as described in WO 2014/124317A1) and ARRY-520(7 mg, 0.017 mmol) in DMF (150 μL) at 0° C. was added HOAt (3.40 mg,0.025 mmol) and HATU (9.49 mg, 0.025 mmol), followed by DIEA (5.82 μL,0.033 mmol) and stirring was continued for 95 minutes.

The reaction was quench by the addition of acetic acid and the crudemixture was purified by C18 RP HPLC to yield Boc protected compound 18(7 mg, 62% yield). ESI MS: C₃₁H₄₀F₂N₆O₇S [M+H] 679.26; found 678.9. TheBoc protecting group was removed by treatment with TFA to give thecompound 18.

Example 4: Synthesis of Compound 19

Compound 19 was prepared as described in Example 3 except2,2-dimethyl-4,9-dioxo-3,11-dioxa-5,8-diazatridecan-13-oic acid (5.86mg, 0.024 mmol) was used instead of2,2-dimethyl-4,9-dioxo-3,11-dioxa-5,8-diazatridecan-13-oic acid. Bocprotected compound 19: ESI MS: C₃₀H₃₈F₂N₆O₆S [M+H]⁺ 649.25; found 648.8.

Example 5: Synthesis of Compound 21

Part A:

To SN38 (4.415 g, 11.25 mmol) in THF (380 mL) at 0° C. was slowly addedHOBt hydrate (1.725 g, 11.25 mmol) in THF (65 mL) followed by thedropwise addition of 4-nitrophenyl carbonochloridate (4.54 g, 22.50mmol) in THF (30 mL) and subsequent addition of DIEA (9.83 mL, 56.6mmol). After 1 hour additional HOBt hydrate (1.725 g, 11.25 mmol) in THF(65 mL), DIEA (9.83 mL, 56.6 mmol) and 4-nitrophenyl carbonochloridate(1.135 g, 5.6 mmol) in THF (10 mL) were added. After 2.5 hours neat2-(methylamino)ethanol (5.02 ml, 62.4 mmol) was added and the resultingreaction mixture was allowed to warm up to room temperature overnight.The crude reaction mixture was concentrated by rotary evaporation andpurified by RP C18 column CombiFlash chromatography using ACN/watercontaining HOAc (0.1%) gradient as eluant, to give compound 20 (3.695 g,71.4% yield). ESI MS: C₂₆H₂₇N₃O₇ [M+H]⁺ 494.18; found 494.0.

Part B:

To Boc-Alanine (0.767 g, 4.05 mmol) in anhydrous DCM (170 mL) at 0° C.,was added EDC (1.554 g, 8.11 mmol) followed by DMAP (0.495 g, 4.05mmol). After 10 minutes compound 20 (2.0 g, 4.05 mmol) in anhydrous DCM(55) and added over 35 minutes. The reaction mixture was allowed to warmup to room temperature slowly over 1-1.5 hours. LC-MS analysis indicatedthe reaction was complete The crude reaction mixture was concentrated byrotary evaporation and purified by RP C18 column CombiFlashchromatography using ACN/water containing HOAc (0.1%) gradient aseluant, to give Boc protected compound 21 (1.70 g, 63% yield). The Bocprotecting group was removed by treatment with TFA to give the compound21 (1.97 g, 51% yield). ESI MS: C₂₉H₃₂N₄O₈ [M+H]⁺ 565.2; found 565.4.

Example 6: Synthesis of Dimeric PEG8 Scaffold (24)

Part A:

The methyl ester of compound 22 (0.495 g, 91% yield) was synthesizedfrom3,3′-((3-((tert-butoxycarbonyl)amino)pentanedioyl)bis(azanediyl))dipropionicacid (105 mg, 0.271 mmol, prepared from Boc protected homoaspartic acidusing the procedures described in Atwell et al, Journal of MedicinalChemistry, 29(1), 69-74; 1986 and WO2004089362) and compound 5 using theprocedure described in Example 2, Part F. ESI MS: C₈₂H₁₄₅N₁₇O₄₀ m/z:[M+2H]²⁺/2 1004.99 found 1004.5. The methyl ester of compound 22 wasremoved by treatment with LiOH using the procedure described in Example1, Part E to give compound 22 (456 mg, 93% yield). ESI MS:C₈₀H₁₄₁N₁₇O₄₀: [M+H] 1980.95; found 1980.9.

Part B:

Boc protected compound 23 was prepared from compound 22 (5.14 mg, 2.59μmol) and compound 18 (3 mg, 5.18 μmol) using the procedure described inExample 2, Part H. ESI MS: C₁₃₂H₂₀₁F₄N₂₉O₄₈S₂: [M+2H]²⁺/2 1551.18; found1550.7. Boc protecting group was removed by treatment with TFA to givethe compound 23.

Part C

Scaffold 24 (3.05 mg) was prepared from compound 23 using the proceduredescribed in Example 2, Part I. ESI MS: C₁₄₁H₂₁₁F₄N₃₁O₅₂S₂: [M+3H]³⁺/31105.17; found 1105.15.

Example 7: Synthesis of Trastuzumab Conjugate of Scaffold 24 (Conjugate25)

To a solution of Trastuzumab (10 mg, 0.067 μmol), in TEAA buffer (50mM), 1 mM EDTA, pH 7, 1.03 mL) was added a solution of TCEP (0.077 mg,0.270 μmol) and the resulting mixture was incubated for 1 h at 37° C.The reaction mixture was allowed to cool to room temperature and thendiluted with TEAA buffer (2.9 mL). A solution of scaffold 24 (2.24 mg,0.676 umol) (prepared as described in Example 6) in DMSO (0.5 mL) wasthen slowly added while vigorously stirring the reaction mixture. Thereaction mixture was stirred at room temperature for ˜1 h beforequenching with cysteine (0.82 mg, 6.77 μmol). The crude product waspurified by SEC (Biosep SEC 3000, pH 5.5, 50 mM sodium phosphate, 300 mMNaCl) followed by buffer exchange into formulation buffer (25 mMcitrate, 75 mM NaCl, 50 mg/mL trehalose, pH 5.5) to give the conjugate25 (3.85 mg, 38.5% yield). Purified conjugate had a drug to trastuzumabratio of about 12.5 as determined by UV-Vis.

Example 8: Synthesis of Dimeric PEG8 Scaffold (27)

Part A:

Boc protected compound 26 was prepared from compound 22 (8.76 mg, 4.42μmol) and compound 19 (4.85 mg, 8.84 μmol) using the procedure describedin Example 2, Part H. ESI MS: C₁₃₀H₁₉₇F₄N₂₉O₄₆S₂ [M+2H]²⁺/2 1521.16found 1521.2. Boc protecting group was removed by treatment with TFA togive the compound 26.

Part B:

Scaffold 27 (4.05 mg) was prepared from compound 26 using the proceduredescribed in Example 2, Part I. ESI MS: C₁₃₉H₂₀₇F₄N₃₁O₅₀S₂m/z:[MH+2H]²⁺/2=1626.7 found 1626.70; [MH+3H]³⁺/3=1084.80 found 1084.81.

Example 9: Synthesis of Trastuzumab Conjugate of Scaffold 27 (Conjugate28)

Conjugate 28 was prepared from scaffold 27 using the procedure describedin Example 7. The purified conjugate had a drug to trastuzumab ratio of2.8.

Example 10: Synthesis of Dimeric PEG8 Scaffold (30)

Part A:

Boc protected compound 29 was prepared from compound 22 (7.5 mg, 3.79μmol)) and ARRY-520 (1.910 mg, 4.54 μmol) using the procedure describedin Example 2, Part H except HATU (7.20 mg, 0.019 mmol) was used insteadof EDC. ESI MS: C₁₂₀H₁₈₁F₄N₂₅O₄₂S₂: [M+2H]²⁺/2 1393.11; found 1392.8.Boc protecting group was removed by treatment with TFA to give thecompound 29.

Part B:

Scaffold 30 (5 mg, 75% yield) was prepared from compound 29 using theprocedure described in Example 2, Part I. ESI MS: C₁₂₉H₁₉₁F₄N₂₇O₄₆S₂ m/z[M+2H]²⁺/2 1498.14; found 1497.8.

Example 11: Synthesis of Trastuzumab Conjugate of Scaffold 30(Conjugates 31A and 31B)

Conjugates 31 A and 31B was prepared from scaffold 30 using theprocedure described in Example 7. Purified conjugate 30A had a drug totrastuzumab ratio of 7.8 and conjugate 30B a drug to trastuzumab ratioof 7.6.

Example 12: Synthesis of Dimeric PEG8 Scaffold (33)

Part A:

Boc protected compound 32 was prepared from compound 22 (12 mg, 6.06μmol)) and compound 21 (13.32 mg, 18 μmol) using the procedure describedin Example 2, Part H. ESI MS: C₁₃₈H₂₀₁N₂₅O₅₄: [M+2H]²⁺/2 1537.19; found1537.3. Boc protecting group was removed by treatment with TFA to givethe compound 32.

Part B:

Scaffold 33 (4.5 mg) was prepared from compound 32 using the proceduredescribed in Example 2, Part I. ESI MS: C₁₄₇H₂₁₁N₂₇O₅₈ [M+2H]²⁺/21642.22; found 1641.8; [M+3H]³⁺/3 1095.17 found 1095.0.

Example 13: Synthesis of Trastuzumab Conjugate of Scaffold 33(Conjugates 34A and 34B)

Conjugates 34 A and 34B was prepared from scaffold 33 using theprocedure described in Example 7. Purified conjugate 34A had a drug totrastuzumab ratio of 6.4 and conjugate 34B a drug to trastuzumab ratioof 6.3.

Example 14: Synthesis of Compound 35

Boc protected compound 35 was prepared by treating Boc-Glu(OtBu)-OH (128mg, 0.422 mmol) in DMF (1.5 mL) at 0° C., with NHS (81 mg, 0.704 mmol)followed by EDC (135 mg, 0.704 mmol). Separately auristatinF-hydroxypropyl amide-alanine TFA-salt (400 mg, 0.352 mmol, prepared asdescribed in U.S. Pat. No. 8,865,383) and DIEA (0.184 mL, 1.056 mmol) inDMF (1 mL) were stirred at 0° C. for 10 min was added. This homogeneousmixture was added to the reaction mixture. The resulting mixture wasstirred at 0° C. for about 1 hour and then at room temperatureovernight. The reaction mixture was concentrated by rotary evaporation,followed by purification by RP HPLC (297 mg, 73% yield). The Bocprotecting group was removed by treatment with TFA to give compound 35(114 mg, 75% yield). ESI MS: C₅₅H₉₄N₈O₁₂ [M+H]⁺ 1059.70 found 1059.4.

Example 15: Synthesis of Compound 36

Compound 36 was prepared as described in Example 14 exceptBoc-Asp(OAll)-OH (80 mg, 0.292 mmol) was used instead ofBoc-Glu(OtBu)-OH. ESI MS (Boc protected compound 36): C₅₈H₉₆N₈O₁₄ [M+H]⁺1129.70; found 1129.6.

Example 16: Synthesis of Compound 37

Compound 37 was prepared as described in Example 14 exceptBoc-Asp(OtBu)-OH (1 g, 0.857 mmol) was used instead of Boc-Glu(OtBu)-OH.ESI MS: C₅₈H₉₆N₈O₁₄ [M+H]⁺ 1129.70; found 1129.6.

Example 17: Synthesis of Glucamine Scaffold 42

Part A:

Boc protected compound 38 was prepared by reacting Boc-Glu-OH (837 mg,2.76 mmol) in DMF (5 mL) at 0° C. with NHS (476 mg, 4.14 mmol) and EDC(794 mg, 4.14 mmol) followed by the addition of D-glucamine (500 mg,2.76 mmol) in 8 mL of DMF (8 mL) and water (2.5 mL) according to theprocedure described in Example 1, Part B. The product was purified by RPC18 column CombiFlash chromatography using ACN/water containing HOAc(0.1%) gradient as eluant (737 mg, 57% yield). ESI MS: C₂₀H₃₈N₂O₁₀[M+H]⁺ 467.25; found 467.3. The Boc protecting group was removed bytreatment with TFA to give compound 38. ESI MS: C₁₅H₃₀N₂O₈ [M+H]⁺ 367.2;found 367.1.

Compound 39 was synthesized from compound 4 (80 mg, 0.143 mmol) andcompound 38 (80 mg, 0.218 mmol) using the procedure described in Example1, Part D (72 mg, 55% yield). ESI MS: C₃₆H₆₂N₈O₁₉ [M+H]⁺ 911.4; found911.3.

Part B:

Compound 40 was synthesized from compound 39 (21 mg, 0.023 mmol) andtrimethylstannanol (29.2 mg, 0.161 mmol) using the procedure describedin Nicolaou, K. C. et al. Angew. Chem. Int. Ed. 44, 1378-1382, 2005,followed by purification by RP C18 column CombiFlash chromatographyusing ACN/water containing HOAc (0.1%) gradient as eluant (15 mg, 72.5%yield). ESI MS: C₃₅H₆₀N₈O₁₉ [M+H]⁺ 897.4; found 897.3.

Part C:

Compound 41 was synthesized from compound 40 (15.0 mg, 0.017 mmol) andauristatin F-hydroxypropyl amide-alanine TFA-salt (19.52 mg, 0.017 mmol,prepared as described in U.S. Pat. No. 8,865,383) using the proceduredescribed in Example 2, Part H (18 mg, 61% yield). ESI MS: C₈₁H₁₃₇N₁₅O₂₇m/z: [M+2H]²⁺/2 877.5; found 877.5.

Part D:

Scaffold 42 was synthesized from compound 41 (18 mg, 10.27 μmol) and2,5-dioxopyrrolidin-1-yl3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1yl)propanamido)ethoxy)ethoxy propanoate (6.39 mg, 0.015 mmol) using the procedure described inExample 2, Part I (7 mg, 53% yield). ESI MS: C₈₆H₁₃₉N₁₇O₃₁ m/z:[M+2H]²⁺/2 953.99; found 953.9.

Example 18: Synthesis of Trastuzumab Conjugate of Scaffold 42(Conjugates 43A and 43B)

Conjugates 43A and 43B were prepared from scaffold 42 using theprocedure described in Example 7. Purified conjugate 43A had a drug totrastuzumab ratio of 6.6 and conjugate 43B a drug to trastuzumab ratioof 6.5.

Example 19: Synthesis of Monomeric PEG8 Scaffold (45)

Part A:

Compound 44 was synthesized from compound 6 (20 mg, 0.022 mmol) andauristatin F-hydroxypropyl amide-alanine TFA-salt (25.5 mg, 0.022 mmol,prepared as described in U.S. Pat. No. 8,865,383) using the proceduredescribed in Example 2, part H (10 mg, 25% yield). ESI MS: C₈₃H₁₄₄N₁₄O₂₇m/z: [M+2H]²⁺/2 885.5 found 885.6.

Part B:

Scaffold 45 was synthesized from compound 44 (9.5 mg, 5.65 μmop and2,5-dioxopyrrolidin-1-yl3-(2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1yl)propanamido)ethoxy)ethoxy propanoate (4.84 mg, 0.011 μmol) using the procedure described inExample 2, Part I (8 mg, 71% yield). ESI MS: C₉₂H₁₅₄N₁₆O₃₁ [M+H]²⁺/2990.55 found 990.6.

Example 20: Synthesis of Trastuzumab Conjugate of Scaffold 45(Conjugates 46A and 46B)

Conjugates 46A and 46B were prepared from scaffold 45 using theprocedure described in Example 7. Purified conjugate 46A had a drug totrastuzumab ratio of 6.8 and conjugate 46B a drug to trastuzumab ratioof 6.5.

Example 21: Synthesis of Trastuzumab Conjugate of Scaffold 47 (Conjugate48)

Scaffold 47 was synthesized as described in Example 19 except that thePEG6 analogue of compound 6 (20 mg, 0.024 mmol) and auristatinF-hydroxypropyl amide-alanine TFA-salt (29.7 mg, 0.025 mmol, prepared asdescribed in U.S. Pat. No. 8,865,383) were used in Example 19, Part A.ESI MS (Compound 47): C₈₈H₁₄₆N₁₆O₂₉ [M+2H]²⁺/2 946.52; found 946.5.

Conjugate 48 was prepared from scaffold 47 using the procedure describedin Example 7. Purified conjugate 48 had a drug to trastuzumab ratio of5.5.

Example 22: Synthesis of Trastuzumab Conjugate of Scaffold 49 (Conjugate50)

Scaffold 49 was synthesized as described in Example 19 except that thePEG12 analogue of compound 6 (101 mg, 0.092 mmol) and compound 36 (95mg, 0.092 mmol) were used in Example 19, Part A. ESI MS (Compound 49):C₁₀₃H₁₇₂N₁₆O₃₅: [M+2H]²⁺/2 1097.6 found 1098.0; [M+Na+H]²⁺/2 1108.6;found 1108.5.

Conjugate 50 was prepared from scaffold 49 using the procedure describedin Example 7.

Example 23: Synthesis of Trastuzumab Conjugate of Scaffold 51 (Conjugate52)

Scaffold 51 was synthesized as described in Example 19 except that thePEG12 analogue of compound 6 (100 mg, 0.092 mmol) and compound 35 (102mg, 0.096 mmol) were used in Example 19, Part A. ESI MS (Compound 51):C₁₀₅H₁₇₇N₁₇O₃₈ [M+2H]⁺/2 1143.8 found 1143.6.

Conjugate 52 was prepared from scaffold 51 using the procedure describedin Example 7. Purified Conjugate 52 had a drug to trastuzumab ratio of12.6.

Example 24: Synthesis of XMT-1535 Conjugates of Scaffold 17 (Conjugates53A, 53B, 53C 53D, 53E)

Conjugates 53A to 53E were prepared from scaffold 17 using the proceduredescribed in Example 7 except that XMT-1535 was used instead ofTrastuzumab and the amount of TCEP used in the reduction of XMT-1535 wasvaried as indicated in Table 1.

TABLE 1 Conjugate TCEP Drug to XMT- # equivalents 1535 ratio 53A 2 10.353B 3 16.4 53C 3 16.9 53D 3 15.2 53E 4 21.8

Example 25: Synthesis of XMT-1535 Conjugates of Scaffold 54 (Conjugates55A, 55B, 55C)

Scaffold 54 was synthesized as described in Example 2 except compound 35(104 mg, 0.068 mmol) were used in Example 2, Part H. ESI MS:C₂₉₇H₅₀₀N₅₄O₁₀₄ m/z: [M+5H]⁵⁺/5=1298.51; found 1298.4;[M+6H]⁶⁺/6=1082.258; found 1082.2; [M+7H]⁷⁺/7=927.793; found 927.8.

Conjugate 55 was prepared from scaffold 54 and XMT-1535 using theprocedure described in Example 7. Purified conjugate 55A had a drug toXMT-1535 ratio of 24.7, conjugate 55B a drug to XMT-1535 ratio of 19.7and conjugate 55C a drug to XMT-1535 ratio of 14.5.

Example 26: Synthesis of XMT-1535 Conjugates of Scaffold 56 (Conjugates57)

Scaffold 56 was synthesized as described in Example 2 except compound 37(138 mg, 0.116 mmol) were used in Example 2, Part H. ESI MS:C₂₉₄H₄₉₄N₅₄O₁₀₄: [M+5H]⁵⁺/5=1290.10 found 1289.7; [M+7H]⁷⁺/7=921.78found 921.8.

Conjugate 57 was prepared from scaffold 56 and XMT-1535 using theprocedure described in Example 7. Purified conjugate 57 had a drug toXMT-1535 ratio of 16.5.

Example 27: Synthesis of XMT-1535 Conjugates of Scaffold 58 (Conjugates59)

Scaffold 58 was synthesized as described in Example 2 except that thePEG12 analogue of compound 15 (152 mg, 0.039 mmol) and compound 35 (176mg, 0.146 mmol) were used in Example 2, Part H. ESI MS (Compound 58):C₃₂₁H₅₄₈N₅₄O₁₁₆ [M] 7015.86; found 7015.86.

Conjugate 59 was prepared from scaffold 58 and XMT-1535 using theprocedure described in Example 7. Purified conjugate 59 had a drug toXMT-1535 ratio of 15.9.

Example 28: Synthesis of XMT-1535 Conjugates of Scaffold 60 (Conjugate61)

Scaffold 60 was synthesized as described in Example 2 except that thePEG12 analogue of compound 15 (170 mg, 0.044 mmol) and auristatinF-hydroxypropyl amide-alanine TFA-salt (179 mg, 0.163 mmol, prepared asdescribed in U.S. Pat. No. 8,865,383) were used in Example 2, Part H.ESI MS: C₃₀₆H₅₂₇N₅₁O₁₀₇ [M] 6628.74; found 6628.75.

Conjugate 61 was prepared from scaffold 60 and XMT-1535 using theprocedure described in Example 7. Purified conjugate 61 had a drug toXMT-1535 ratio of 16.3.

Example 29: Synthesis of XMT-1535 Conjugates of Scaffold 65 (Conjugate66)

Part A:

Compound 64 was prepared by reacting compound 62 (5.03 mg, 5.71 μmol,prepared as described in Example 2, Part E, except dimethyl3,3′-(2-amino-2-((3-methoxy-3-oxopropoxy)methyl)propane-1,3-diyl)bis(oxy)dipropanoatewas used instead of compound 10) with compound 63 (40 mg, 0.019 mmol,prepared by reaction compound 6 (100 mg, 0.092 mmol) with compound 36(153 mg, 0.135 mmol) using the procedure described in Example 2, Part H.ESI MS (Compound 64): C₃₁₆H₅₄₁N₅₁O₁₁₁ [M] 6826.83; found 6826.842.

Scaffold 65 was synthesized as described in Example 2, Part I. ESI MS(scaffold 65): C₃₁₆H₅₃₉N₅₃O₁₁₅ [M] 6916.795; found 6916.752.

Conjugate 66 was prepared from scaffold 58 and XMT-1535 using theprocedure described in Example 7. Purified conjugate 66 had a drug toXMT-1535 ratio of 15.

Example 30: Synthesis of Trastuzumab Conjugate of Scaffold 54 (Conjugate67)

Conjugate 67 was prepared from scaffold 54 and Trastuzumab using theprocedure described in Example 7. Purified conjugate 61 had a drug toXMT-1535 ratio of 13.0.

Example 31: Synthesis of Trastuzumab Conjugate of Scaffold 58 (Conjugate68)

Conjugate 68 was prepared from scaffold 58 and Trastuzumab using theprocedure described in Example 7. Purified conjugate 68 had a drug toXMT-1535 ratio of 12.1

Example 32: Synthesis of Bis-Glucamine Scaffold 75

Part A:

To 2-(((benzyloxy)carbonyl)amino)pentanedioic acid (2 g, 7.11 mmol) inwater (75 mL) was added potassium carbonate (0.491 g, 3.56 mmol). To theresulting mixture at 0° C. was added HOAt (2.420 g, 17.78 mmol) and EDC(4.09 g, 21.33 mmol). After 10-15 min. D-glucamine (2.96 g, 16.35 mmol)in water (20 mL) was added followed by additional potassium carbonate(0.491 g, 3.56 mmol) in water (1 mL) until pH˜7. After 24 hours, LC-MSindicated the reaction was incomplete. Additional HOAt (500 mg),D-glucamine (0.9 g) in water (3 mL) and EDC (1.5 g) were added and pH ofthe resulting mixture adjusted to 7-8 with K₂CO₃ (250 mg). The reactionmixture concentrated to dryness, dissolved in 10% MeOH in DCM was firstpurified by SiO₂ column chromatograph using EtOAc/DCM 10-50% as eluantfollowed by further purification to give compound 69 (2.02 g, 47%yield). ESI MS: C₂₅H₄₂N₃O₁₄ [M+H]:=608.27; found 608.3.

Compound 69 (2.015 g, 3.32 mmol) was treated with 10% Pd/C (0.35 g,0.332 mmol) under H₂ as described in Example 1, Part C to give crudecompound 70 quantitatively. ESI MS: C₁₇H₃₆N₃O₁₂ [M+H]:=474.2; found474.3.

Part B:

Compound 4 (750 mg, 1.333 mmol) in DMF was reacted with compound 70 (574mg, 1.212 mmol) at 0° C. using the procedure described in Example 1,Part D to give compound 71 (559 mg, 45% yield). ESI MS: C₃₈H₆₈N₉O₂₃[M+H]⁺ 1018.4, found 1018.3.

Part C:

Compound 71 (555 mg, 0.545 mmol) in water at 0° C. and treated with atotal of LiOH (408 μL of 2 M solution) over 2 hrs. followed by quenchingthe reaction with HOAc (35 μL). The crude product was purified by RP C18column CombiFlash chromatography using ACN/water containing HOAc (0.1%)gradient as eluant, to give compound 72 (333 mg, 61% yield). ESI MS:C₃₇H₆₆N₉O₂₃ [M+H]⁺ 1004.4, found 1004.4.

Part D:

Compound 73 was synthesized from compound 72 (175.0 mg, 0.174 mmol) andauristatin F-hydroxypropyl amide-alanine TFA-salt (168 mg, 0.192 mmol,using the procedure described in Example 2, Part H (67 mg, 21% yield).ESI MS: C₈₃H₁₄₂N₁₆O₃₁ m/z: [M+2H]²⁺/2 930.5; found 930.5.

Part E:

Compound 74 was prepared by reacting compound 13 (5.72 mg, 6.10 μmol5.72 mg, 6.10 μmol), with deprotected compound 73 (40 mg, 0.021 mmol)using the procedure described in Example 2, Part H. ESI HRMS (Compound74): C₂₇₃H₄₆₃N₅₅O₁₀₃ [M] 6160.3; found 6160.3.

Scaffold 75 was synthesized from compound 74 as described in Example 2,Part I. ESI MS (scaffold 75): C₂₈₂H₄₇₃N₅₇O₁₀₇ [M] 6370.33; found6370.35.

Example 33: Synthesis of XMT-1535 Conjugate of Scaffold 75 (Conjugate76)

Conjugate 76 was prepared from scaffold 75 and XMT-1535 using theprocedure described in Example 7 except the TCEP to antibody ratio was3.25. Purified conjugate 76 had a drug to XMT-1535 ratio of 9.3.

Example 34: Synthesis of XMT-1535 Conjugates of Scaffold 77 (Conjugates78)

Scaffold 77 was synthesized as described in Example 32 except thatbenzyl protected compound 35 (259 mg, 0.214 mmol) was used in Example32, Part D. ESI MS (Scaffold 77 MK2-079): C₂₉₇H₄₉₄N₆₀O₁₁₆ m/z:[M+6H]⁶⁺/6 1127.20 found 1127.24.

Conjugate 78 was prepared from scaffold 75 and XMT-1535 using theprocedure described in Example 7 except the TCEP to antibody ratio was3.25. Purified conjugate 78 had a drug to XMT-1535 ratio of 16.3.

Example 35: Synthesis of XMT-1535 Conjugates 79A and Conjugate 79B

Conjugates 79A and 79B were prepared as described in Example 25 exceptthe TCEP to antibody ratio was 4.0. Purified conjugate 79A had a drug toXMT-1535 ratio of 15.5 and purified conjugate 79B had a drug to XMT-1535ratio of 13.7.

Example 36: Synthesis of Bis-Glucamine Scaffold 75

Part A:

The Cbz-protected analogue of compound 13 (93 mg, 0.096 mmol)) inNMP/water (1:2) 2.0 mL was reacted with deprotected compound 71 in theform of its HCl-salt (291 mg, 0.317 mmol) as described in Example 2,Part F to give compound 80 (276 mg, 78% yield). ESI MS: C₁₄₁H₂₃₇N₃₄O₇₉[M+H]⁺ 3670.56; found 3670.25.

Part B:

Compound 80 (175 mg, 0.048 mmol) in 4.9 mL of water (4.9 mL) at 0° C.was treated with aqueous LiOH (1 M solution, 6 equivalents total) togive the hydrolyzed acid compound. The crude product was purified by RPC18 column CombiFlash chromatography using ACN/water containing HOAc(0.1%) gradient as eluant, to give compound 81 (105 mg, 35% yield). ESIMS: C₁₃₈H₂₃₁N₃₄O₇₉ [M+H]⁺ 3628.51; found 3628.57.

Part C:

CBz protected compound 82 was synthesized from compound 81 (88 mg, 0.024mmol) and auristatin F-hydroxypropyl amide-alanine TFA-salt (84 mg,0.085 mmol, using the procedure described in Example 2, Part H (40.5 mg,27% yield). ESI MS: C₂₇₆H₄₆₂N₅₅O₁₀₃ [M+H]⁺: 6195.26; found 6195.37.

The protecting group was removed using 10% Pd/C (198 mg) in EtOH/water(1:1) under H₂ as described in Example 2, Part B, to give compound 82(10 mg, 43% yield). ESI MS: C₂₆₈H₄₅₆N₅₅O₁₀₁ [M+H]⁺: 6061.23; found6061.27.

Part D:

Scaffold 75 was synthesized from compound 82 and as described in Example2, Part I and purified using a dialysis cell equipped with a 1 kDamembrane (yield 82%). ESI MS (scaffold 75): C₂₈₂H₄₇₃N₅₇O₁₀₇ [M] 6370.33;found 6370.50.

Example 37: Synthesis of XMT-1535 Conjugates of Scaffold 75 (Conjugate83)

Conjugate 83 was prepared from scaffold 75 (as prepared in Example 36)and XMT-1535 using the procedure described in Example 7 except the TCEPto antibody ratio was 4.0. Purified conjugate 83 had a drug to XMT-1535ratio of 15.7

Example 38: Synthesis of XMT-1535 Conjugates of Scaffold 84 (Conjugate85)

Scaffold 84 was prepared as described in Example 36, except the benzylderivative of compound 35 (13.97 mg, 0.012 mmol) was used in Part C. ESIMS (scaffold 84): C₂₉₇H₄₉₄N₆₀O₁₁₆ m/z: [M+6H]⁶⁺ 1127.25; found 1127.20.

Conjugate 85 was prepared from scaffold 84 and XMT-1535 using theprocedure described in Example 7 except the TCEP to antibody ratio was4.0. Purified conjugate 85 had a drug to XMT-1535 ratio of 13.1.

Example 39: Cell Viability Assay for the PBRM-Drug Conjugates

The conjugates were evaluated for their antiproliferation properties intumor cell lines in vitro using CellTiter-Glo® (Promega Corp). Cellswere plated at a density of 5,000 cells per well in black walled 96-wellplate and allowed to adhere overnight at 37° C. in a humidifiedatmosphere of 5% CO₂. BT474, SKBR3, NCI-N87 cells (HER2 expressingcells), JIMT1 cells (HER2 medium expression level cells), MCF7 cells(HER2 low expressing levels cells), and OVCAR3 (ovarian adenocarcinomacell line, not amplified) and were plated. CellTiter-Glo® reagent wasadded to the wells at room temperature and the luminescent signal wasmeasured after 10 min using a SpectraMax M5 plate reader (MolecularDevices). Dose response curves were generated using SoftMax Prosoftware. IC₅₀ values were determined from four-parameter curve fitting.

Table I and Table II give illustrative results for the antiproliferationproperties of the PBRM-drug conjugates.

TABLE I BT474 SKBR3 N87 JIMT1 MCF7 Conjugate IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀No. (nmol/L) (nmol/L) (nmol/L) (nmol/L) (nmol/L) 50 0.309 0.04 0.3414.84 1.19 52 0.18 0.02 0.20 15.82 0.14 46A 0.1 0.02 0.07 14.56 0.14 46B0.11 0.03 0.11 100 0.13 43A 0.09 0.02 0.1 17.69 0.14 43B 0.1 0.01 0.0413.53 0.15 48 0.13 0.02 0.09 18.47 0.17 34A 29.14 11.06 22.07 27.7 21.9134B 11.73 9.962 29.39 17.97 20.99 31A <100 <100 <100 <100 <100 31B <100<100 <100 <100 <100 25 <300 <300 <300 <300 <300 28 <300 <300 <300 <300<300

TABLE II Conjugate OVCAR3 IC₅₀ No. (nmol/L) 66 0.03 53A 0.01 53B 0.0153E 0.01 53C 0.03 53D 0.02 55A 0.05 55C 0.03 78 0.015 79A 0.015 83 0.03

As shown in Tables I and II, the PBRM-drug conjugates show efficacy inthe tested cell lines.

Example 40: Tumor Growth Response to Administration of PBRM-DrugConjugates

Female CB-17 SCID mice were inoculated subcutaneously with NCI-N87 cells(n=10 for each group). Test compound or vehicle were dosed IV as asingle dose on day 1. Tumor size was measured at the times indicated inFIG. 1 using digital calipers. Tumor volume was calculated and was usedto determine the delay in tumor growth. Tumor volumes are reported asthe mean±SEM for each group. FIG. 1 provides the results for the tumorresponse in mice inoculated subcutaneously with NCI-N87 cells (n=10 foreach group) after IV administration as a single dose on day 1 of vehicleand Trastuzumab-drug conjugate, Conjugate 43B, Example 18, at 1.5 mg/kg.The results show that on day 70 Conjugate 43B resulted in 100% objectiveregressions consisting of 10 complete responses.

Female CB-17 SCID mice were subcutaneously implanted with OVCAR-3 (n=10for each group). Test compounds or vehicle were dosed IV as a singledose on day 1. Tumor size was measured at the times indicated in FIGS. 2and 3 using digital calipers. Tumor volume was calculated and was usedto determine the delay in tumor growth. Mice were sacrificed when tumorsreached a size of 1000 mm³. Tumor volumes are reported as the mean±SEMfor each group.

FIG. 2 provides the results for the tumor response in micesubcutaneously implanted with OVCAR-3 tumor fragments (n=10 for eachgroup) after IV administration of vehicle; and the XMT-1535-drugconjugates: Example 24, Conjugate 53D; Example 25, Conjugate 55A;Example 29, Conjugate 66; each at 3 mg/kg as a single dose at day 1. Atday 64, conjugates 53D had 6 partial response, 3 complete responses and1 tumor free survivor; Conjugate 66 had 1 partial response, 9 completeresponses and 5 tumor free survivors; and Conjugate 55A had 10 completeresponses and 8 tumor free survivors.

FIG. 3 provides the results for the tumor response in micesubcutaneously implanted with OVCAR-3 tumor fragments (n=10 for eachgroup) after IV administration of vehicle; and the XMT-1535-drugconjugates: Example 25, Conjugate 55C had 6 partial responses, 4complete responses and 1 tumor free survivor; Example 26, Conjugate 57had 4 partial responses, 6 complete responses and 5 tumor freesurvivors; Example 28, Conjugate 61 had 2 partial responses; and Example27, Conjugate 59 had 7 partial responses and 1 complete response; eachat 3 mg/kg as a single dose at day 1.

FIG. 5 provides the results for the tumor response in micesubcutaneously implanted with OVCAR-3 tumor fragments (n=10 for eachgroup) after IV administration of vehicle; and the XMT-1535-drugconjugates: Example 33, Conjugate 76 had 1 partial response, 9 completeresponses and 4 tumor free survivors; Example 26, Conjugate 57 had 1partial response, 8 complete responses and 7 tumor free survivors;Example 25, Conjugate 55C had 2 partial responses, 8 complete responsesand 5 tumor free survivors; each at 3 mg/kg as a single dose at day 1and Example 27, Conjugate 59 at 3.72 mg/kg as a single dose at day 1 had1 partial response, 6 complete responses and 2 tumor free survivors.

FIG. 6 provides the results for the tumor response in micesubcutaneously implanted with OVCAR-3 tumor fragments (n=10 for eachgroup) after IV administration of vehicle; and the XMT-1535-drugconjugates: Example 26, Conjugate 57; Example 33, Conjugate 76; andExample 34, Conjugate 78; each at 1.5 mg/kg as a single dose at day 1and Example 34, Conjugate 78; at 3.0 mg/kg as a single dose at day 1 had4 partial responses, 6 complete responses and 3 tumor free survivors.

Female CB-17 SCID mice were subcutaneously implanted with Calu-3 cells(n=10 for each group). Test compounds or vehicle were dosed IV as asingle dose on day 1. Tumor size was measured at the times indicated inFIG. 4 using digital calipers. Tumor volume was calculated and was usedto determine the delay in tumor growth. Tumor volumes are reported asthe mean±SEM for each group.

FIG. 4 provides the results for the tumor response in micesubcutaneously implanted with Calu-3 cells (n=10 for each group) afterIV administration of vehicle; and the Trastuzumab-drug conjugates:Example 30, Conjugate 67; Example 31, Conjugate 68; each at 1 mg/kg as asingle dose at day 1.

Example 41: Mouse Plasma PK after Administration of PBRM-Polymer-DrugConjugates

Female CD-1 mice were allowed to acclimate for at least 4 days prior toinitial dosing. All mice were given regular chow and water ad libitumand were not fasted prior to compound administration. Test compounds orvehicle were dosed IV as a single dose on day 1.

The mice were injected intravenously with vehicle (n=3) or withPBRM-polymer-drug conjugate, Example 35, Conjugate 79A; Example 35,Conjugate 7B; Example 37, Conjugate 83; and Example 38, Conjugate 85,each at 3 mg/kg, n=27 for each group). Plasma was collected at 5 min, 1h, 3 h, 6 h, 24 h, 48 h, 72 h, day 7 and day 14 post dosing. Body weightwas measured prior to dosing on day 1 and on days 1, 7 and 14. Allanimals were observed throughout the fourteen day period for mortalityor morbidity.

The total AF-HPA (conjugated AF-HPA and unconjugated (free) drug i.e.AF-HPA and AF) concentrations and conjugated AF-HPA concentrations weredetermined by LC-MS/MS analysis.

Table III gives the plasma pK. FIG. 7A shows the total antibodyconcentration; FIG. 7B shows the total AF-HPA concentration and FIG. 7Cshows the conjugated AF-HPA concentration for the PBRM-polymer-drugconjugate.

TABLE III Plasma PK (PBRM-polymer-drug conjugates) AF-HPA to T_(1/2)AUC_(0 to 336) Test Sample Antibody ratio (hr) (μg · hr/mL) C2344 13.7160 557 Example 35, Conjugate 79B C2345 13.1 134 462 Example 38,Conjugate 85 C2262 15.5 202 718 Example 35, Conjugate 79A C2269 15.7 114402 Example 37, Conjugate 83

The results in Table III showed that the PBRM-polymer-drug conjugateshad a half-life of ˜114-202 hours (˜4.75-8.42 days) with anAUC_(0 to 336) of ˜402 to 718 μg·hr/mL and were independent of theAF-HPA to antibody ratio of the PBRM-polymer-drug conjugates.

All publications, including, e.g., non-patent literature, patentapplications, and patents, cited in this specification are incorporatedherein by reference for all purposes. The invention can be embodied inother specific forms without departing from the spirit or essentialcharacteristics thereof. The foregoing embodiments are therefore to beconsidered in all respects illustrative rather than limiting on theinvention described herein. Scope of the invention is thus indicated bythe appended claims rather than by the foregoing description, and allchanges that come within the meaning and range of equivalency of theclaims are intended to be embraced therein.

What is claimed is:
 1. A conjugate selected from the group consistingof:

wherein d₁₃ is 4 or 5; PBRM is a protein based recognition-molecule; andXMT-1535 comprises a variable light chain complementarity determiningregion 1 (CDRL1) comprising the amino acid sequence SASQDIGNFLN (SEQ IDNO: 8); a variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence YTSSLYS (SEQ ID NO: 9); avariable light chain complementarity determining region 3 (CDRL3)comprising the amino acid sequence QQYSKLPLT (SEQ ID NO: 10); a variableheavy chain complementarity determining region 1 (CDRH1) comprising theamino acid sequence GYTFTGYNIH (SEQ ID NO: 5); a variable heavy chaincomplementarity determining region 2 (CDRH2) comprising the amino acidsequence AIYPGNGDTSYKQKFRG (SEQ ID NO: 6); and a variable heavy chaincomplementarity determining region 3 (CDRH3) comprising the amino acidsequence GETARATFAY (SEQ ID NO: 7).
 2. A conjugate of Formula (XXX):

wherein d₁₃ is 4 or 5; PBRM is a protein based recognition-molecule; andeach R_(A) is

wherein: each occurrence of D is independently a therapeutic agenthaving a molecular weight≤about 5 kDa; and each occurrence of L^(D) isindependently a divalent linker moiety connecting D to the peptidemoiety and comprises at least one cleavable bond such that when the bondis broken, D is released in an active form for its intended therapeuticeffect.
 3. The conjugate of claim 2, wherein each R_(A) is


4. The conjugate of claim 2, wherein each R_(A) is


5. A pharmaceutical composition comprising a conjugate of claim 2 and apharmaceutically acceptable carrier.
 6. The conjugate of claim 2,wherein each R_(A) is


7. The conjugate of claim 2, wherein each R_(A) is


8. A conjugate being of Formula (XXX):

wherein d₁₃ is 4 or 5; PBRM is a protein based recognition-molecule; andeach R_(A) is